Glucosylceramide Synthase and Its Functional Interaction with RTN-1C Regulate Chemotherapeutic-Induced Apoptosis in Neuroepithelioma Cells1

[CANCER RESEARCH 63, 3860–3865, July 15, 2003] Advances in Brief

Glucosylceramide Synthase and Its Functional Interaction with RTN-1C Regulate Chemotherapeutic-induced Apoptosis in Neuroepithelioma Cells1

Federica Di Sano,2 Barbara Fazi,2 Gennaro Citro, Penny E. Lovat, Gianni Cesareni, and Mauro Piacentini3 Department of Biology, University of Rome “Tor Vergata,” 00133 Rome, Italy [F. D., B. F., G. Ce., M. P.]; Istituto Regina Elena, 00156 Rome, Italy [G. Ci.]; Northern Institute of Cancer Research and School of Clinical Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom [P. E. L.]; and Cell Biology and E.M. Unit, INMI, IRCCS “Lazzaro Spallanzani,” Rome, Italy [M. P.]

Abstract treatment of cells with different GCS inhibitors affects basic cellular functions, including growth, death, and adhesion (10). Glucosylceramide synthase (GCS), the key enzyme in the biosynthesis Recent studies have demonstrated a direct correlation between the of glycosphingolipids, has been implicated in many biological phenomena, development of multidrug resistance and increased levels of GC (11, including multidrug resistance. GCS inhibition, by both antisense and the 12), hence, GCS has been suggested as a candidate target for cancer specific inhibitor (D-threo)-1-phenyl-2-decanoylamino-3-morpholino-1- propanol (PDMP), results in a drastic decrease of apoptosis induced by the therapy. However, it remains controversial as to how the inhibition of p53-independent chemotherapeutic agent N-(4-hydroxyphenyl)retinamide GCS could represent a way to sensitize transformed cells to chemo- in neuroepithelioma cells. By using the yeast two-hybrid system, we have therapeutic agents (13). In fact, in previous studies, we reported that identified a member of the reticulon (RTN) family (RTN-1C) as the major the apoptotic response of GCS antisense clones to various p53- GCS-protein partner. Interestingly, RTN-1C not only interacts with GCS dependent anticancer drugs (doxorubicin, etoposide, and cisplatin) at Golgi/ER interface but also modulates its catalytic activity in situ.In was not increased (14). We used the synthetic retinoid fenretinide to fact, overexpression of RTN-1C sensitizes CHP-100 cells to fenretinide- elucidate whether GCS might be involved in a p53-independent induced apoptosis. These findings demonstrate a novel p53-independent drug-induced apoptosis pathway. In fact, fenretinide has been shown pathway of apoptosis regulated by Golgi/endoplasmic reticulum protein to induce apoptosis in a variety of cancer cell lines (15, 16) through interactions, which is relevant for cancer combined therapy. a p53-independent pathway involving the induction of ER-response gene GADD153 and the generation of reactive oxygen species (17). Introduction We show here that GCS is involved in fenretinide-dependent apo- GCS4 catalyzes the first glycosylation step in the biosynthesis of ptosis of cancer cells and its action is specifically modulated by the GSLs by transferring the glucose from UDP-glucose to ceramide (1, interaction with a member of the RTN family, RTN-1C. These results 2). After its translocation to the Golgi lumen, GC can be additionally suggest that the p53-independent pathway of fenretinide-induced ap- metabolized to higher GSLs, which are major constituents of the outer optosis is regulated by the GCS/RTN-1C complex at the ER/Golgi leaflet of the plasma membrane in eukaryotic cells. GSLs play an interface. essential role in many biological processes, including development, cell death, tumor progression, and pathogen/host interaction (3–5). Materials and Methods GCS is a type III integral protein, localized in the cis/medial Golgi, Materials. CHP-100 human neuroepithelioma were kindly donated by which has a single membrane spanning region near its NH2 terminus, Gerry Melino (University of Rome “Tor Vergata,” Italy). C6-NBD was from whereas most of the protein, including the catalytic site, faces the Molecular Probes (Eugene, OR). PDMP and the anti-␤-tubulin mouse mono- cytoplasm (2, 6). clonal antibody were from Sigma Chemical Company (St. Louis, MO). 4-HPR Although previous studies have suggested different ways by which was from Janssen-Cilag Ltd. (Saunderton, United Kingdom) as described GCS is regulated (7, 8), it is not clear how the enzyme is modulated previously (17). The anti-GFP rabbit polyclonal antibody was from Santa Cruz in the Golgi apparatus and consequently the molecular mechanism(s) Biotechnology, Inc. (Santa Cruz, CA). The anti-RFP rabbit polyclonal anti- at the basis of its pleiotropic effect on cellular functions. body was from Clontech (Palo Alto, CA). The anti-calnexin and anti-membrin Compelling evidence has been presented indicating that GCS is antibodies were from Stressgene (Victoria, British Columbia). The anti-␤-CopI constitutively expressed in a variety of tissues (6) and that the syn- rabbit polyclonal antibody was from Vinci-Biochem (Lausen, Switzerland). thesis of GSLs is vital for embryonic development (9). Furthermore, The chemiluminescence ECL detection system was from Amersham Corp. (Burck, United Kingdom). High-performance thin layer chromatography slice gel 60 plates were from Merck (Darmstadt, Germany). Received 3/10/03; accepted 5/13/03. Antibodies. Peptides with the sequence corresponding to amino acid 5–14 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with of human GCS or amino acid 6–20 of human RTN-1C were synthesized and 18 U.S.C. Section 1734 solely to indicate this fact. used as antigens for immunization in rabbits. To generate specific antisera 1 The work was partially supported by grants from European Community “Apoptosis peptides were covalently linked to the carrier protein cBSA (Pierce, Rockford, Mechanisms,” Associazione Italiana Ricerca sul Cancro and AIDS project from Ministero IL). After 5 weekly inoculations with the immunogen the antisera fram rabbits della Salute (to M. P.) and Progetto Finalizzato 2002, from Ministero della Salute (to G. Ci.), and from an Associazione Italiana Ricerca sul Cancro grant (to G. Ci.). F. D. was were tested and then the antibodies were purified using a protein A-Sepharose partially supported by a fellowship by Federazione Italiana Ricerca Cancro. P. E. L. was CL-4B column (Amersham, Burck, United Kingdom). supported by CLIC United Kingdom. Yeast Two-Hybrid System. The yeast two-hybrid screen was performed 2 The first two authors contributed equally to this article. 3 with yeast strain PJ69–4A MATa trp1–901 leu2–3, 112 ura3–52 his3–200 To whom requests for reprints should be addressed, at Department of Biology, ⌬ ⌬ University of Rome “Tor Vergata,” Via della Ricerca Scientifica, 00133 Rome, Italy. gal4 gal80 LYS2::GAL1-HIS3 GAL2-ADE2 mt2::GAL7-lacZ (18). The Phone: 003906-72594370; Fax: 003906-2023500; E-mail: [email protected]. plasmid used as bait was constructed by fusion of GAL4BD to human GCS 4 The abbreviations used are: GCS, glucosylceramide synthase; GC, glucosylceramide; coding sequence into the EcoRI site of the vector p21.29 AmpR ColE1ORI, GSL, glycosphingolipid; RTN, reticulon; ER, endoplasmic reticulum; C6-NBD, 6-[N- TRP1, CEN6, GAL4BD (amino acid 1–147). The human brain cDNA library (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoylsphingosine; PDMP, (D-threo)-1- phenyl-2-decanoylamino-3-morpholino-1-propanol; Fenretinide, N-(4-hydroxyphenyl) was cloned into pACT2 vector AmpR ColE1ORI, LEU2, GAL4AD (amino retinamide; GFP, green fluorescent protein; RFP, red fluorescent protein. acid 768–881; Clontech). The positive clones were selected on synthetic 3860

medium lacking tryptophan, leucine, adenine, and histidine. Healthy colonies AS) were treated with fenretinide for 24 h, and apoptosis was evalu- that also displayed ␤-galactosidase activity were isolated and sequenced. ated by flow cytometric analysis (Fig. 1A). Interestingly, we detected GCS and RTN-1C Expression Vectors and Transfection. The full length a decrease of the apoptotic response to fenretinide in GCS antisense of human GCS, RTN-1C, or RTN-3 were obtained from cDNA human brain cells, paralleled by a drastic reduction of the expression of GADD153 library by PCR amplification using specific primers. Fragments were cloned (Fig. 1B), which is known to mediate fenretinide-dependent apoptosis into BglII/BamHI sites of pDsRed1-N1 or HindIII/BamHI sites of pEGFP-C1 (17). To confirm the involvement of the enzyme activity in this vectors (Clontech, Palo Alto, CA), respectively. CHP-100 were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) as described previously (14). CHP-100 pathway, CHP-100 cells were treated with fenretinide in the presence cells (80% confluent) were transiently transfected by lipofection according to or absence of PDMP, a specific inhibitor of GCS (Fig. 1C). As the manufacture’s specifications (DMRIE-C Transfection Reagent; Invitrogen, suggested by the antisense approach, the apoptotic response to the United Kingdom). retinoid was also suppressed by the inhibition of GCS activity, thus Immunoprecipitation and Western Blotting. Cells were washed twice demonstrating that the functional enzyme is essential for fenretinide- with PBS and scraped into RIPA assay buffer [150 mM NaCl, 1% NP40, 0.5% induced apoptosis. These results are particularly interesting consider- sodium deoxycholate, 0,1% SDS, 50 mM Tris-HCl (pH 7.5)] with freshly ing that GCS is not involved in the p53-mediated apoptosis induced added protease inhibitors. After an incubation for 30 min on ice and a brief by chemotherapeutic drugs (14). In fact, fenretinide-induced apoptosis sonication, the lysate was centrifuged at 14,000 rpm for 10 min at 4°C to occurs via a p53-independent pathway and its effect is synergistic remove the insoluble cell debris. The anti-GCS antibody was coupled to with that of DNA-damaging agents (15). Nevertheless the precise Dynabeads protein G (Dynal, Oslo, Norway) using dimethylpimelimidate coupling according to the manufacture’s instructions. Equal amounts of cel- mechanism of action of fenretinide is presently not clear. lular protein were incubated for 2 h with antibody-linked beads at 4°C with In the attempt to better understand the regulation of GCS, we continuous rocking. After washing in PBS buffer, the beads were boiled in searched for potential GCS interactors by the yeast two-hybrid ap- SDS buffer, and samples were resolved by 12% SDS-PAGE, transferred proach using the full-length human GCS as bait to screen a human overnight at 25 mA onto nitrocellulose paper, and analyzed by Western adult brain library. We analyzed 6 ϫ 106 library clones and, among blotting as described previously (19). the 160 clones that were able to grow on synthetic medium in the Immunocytochemistry. Cells were fixed in 4% paraformaldehyde for absence of histidine and adenine, 13 were positive for ␤-galactosidase 20 min at room temperature and permeabilized with 0.1% Triton-1X-100 in staining. Sequence analysis of these 13 clones resulted in the identi- PBS for 20 min at room temperature. Cells were then washed with PBS, and nonspecific binding was blocked by incubating the cells with 5% BSA in PBS for 30 min at room temperature. Indirect immunofluorescence was performed incubating the cells with anti-␤-CopI, diluted 1:2000 in 1% BSA (blocking solution) for1hatroom temperature. Washes were followed by incubation with the antirabbit antibody (Alexa-Fluor, Burlingame, CA, 568 diluted 1:1000 in blocking solution for1hatroom temperature). Fluorescence was then evaluated with a confocal microscope (Nikon Instruments Spa, Eclipse TE200) equipped with EZ2000 software for PCM2000. ␮ GCS Assay. Cells were incubated with 5 M C6-NBD for 2 h, and total lipids were extracted from cells and culture medium according to the method of Bligh and Dyer (20) After partition, the chloroformic phase was collected and lipids resolved by High-performance thin layer chromatography in chlo-

roform/methanol/water (65:25:4, vol/vol). GlcC6-NBD was detected under UV illumination, extracted from the silica-gel in 3 ml of ethanol and its fluorescence measured by a Perkin-Elmer LS-5 luminescence spectrometer. Quanti-

tation was carried out referring to a GlcC6-NBD fluorescence calibration curve. Apoptosis Evaluation by Flow Cytometric Analysis. Cells were detached by trypsinization and centrifuged at 300 ϫ g for 5 min; pellets were washed with PBS (pH 7.4), placed on ice, and fixed with ice-cold 70% ethanol for 1 h. After washing in PBS, pellets were incubated with 100 ␮g/ml RNAsi (Roche, Basel, Switzerland) for 20 min at room temperature and stained with propidium iodide (50 ␮g/ml) at 4°C for 30 min, before analysis by flow cytometry using a FACScan Flow Cytometer (Becton-Dickinson, Franklin Lakes, NJ) at 565 and 605 nm as described previously (21).

Results and Discussion

It has recently been shown that fenretinide-induced apoptosis of cancer cells requires the induction of the ER-stress response gene GADD153 (15–17, 22). It has also been proposed that ceramide metabolism may play a role in this pathway because fenretinide leads to the accumulation of high levels of ceramide from de novo synthesis (16). Considering the role of GCS in multidrug resistance, we inves- Fig. 1. Effect of GCS inhibition on fenretinide-induced apoptosis. A, CHP-100 cells, vector control cells (CHP-Neo), or antisense-GCS cells (CHP-AS) were treated with 12 tigated the possibility that the enzyme may be involved in fenretinide- ␮M fenretinide (FenR) or ethanol control (ctr) for 24 h, fixed, and stained with propidium induced apoptosis. We first analyzed the effect of GCS inhibition (by iodide and analyzed by flow cytometry. Results are means Ϯ SD of three independent ␤ using GCS antisense cells) on the apoptotic response elicited by determinations. B, Western blot analysis of GADD153 (top panel)or -tubulin (bottom panel) expression in CHP-100, CHP-Neo, or CHP-AS cells treated with 12 ␮M fenretinide fenretinide in CHP-100 neuroepithelioma cells. Our CHP-100-derived (FenR) or ethanol control (ctr) for 24 h. C, cells were treated with 12 ␮M fenretinide GCS antisense cells display a reduction of GCS expression resulting (FenR) or ethanol control (ctr) for 24 h in the presence or absence of 20 ␮M PDMP, fixed, ϳ and stained with propidium iodide and analyzed by flow cytometry. Results are (statistically significant (P Ͻ 0.001 ,ء .in a decrease of its specific activity of 60% (14). Control cells means Ϯ SD of three independent determinations (wild-type and CHP-Neo) and GCS antisense-expressing cells (CHP- compared with control cells. 3861

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. RTN-1C MODULATES GCS ACTIVITY IN CANCER CELLS fication of RTN family members as the only GCS-interacting pro- interaction with GCS is very specific and restricted to RTN-1C. To teins; 12 clones were identified as RTN-1C (four different clusters; verify the specificity of this interaction, an unrelated integral mem- Fig. 2, A and B) and 1 as RTN-3. RTNs are neuroendocrine-specific brane protein of the Golgi apparatus (Membrin; Ref. 26) and a proteins encoded by genes that are known to produce different prod- resident ER transmembrane protein (Calnexin; Ref. 27) were also ucts by alternative splicing (23, 24). In particular, the RTN-1 gene tested. Although, these two proteins were detected in the cell lysate, encodes three proteins (RTN-1A, RTN-1B, and RTN-1C) with a they were not present in the GCS/RTN-1C complexes (Fig. 3A). conserved COOH-terminal region (ϳ70% identical with the other These results confirm and extend the yeast two-hybrid findings indi- members of RTN family) containing two short hydrophobic domains cating that RTN-1C specifically interacts with GCS in mammalian that are predicted to be membrane spanning domains. In fact, RTN-1 cells. is integral membrane proteins tightly associated with the ER mem- We additionally characterized the GCS/RTN-1C interaction by brane (Ref. 25; Fig. 2, C and D). All of the RTN clones identified analyzing their intracellular localization. To this aim, NH2-terminal contain the lumenal loop and the conserved COOH-terminal tail, GFP-tagged RTN-1C and RFP-tagged GCS expression plasmids were localized in the cytoplasm; thus, it is likely that these two regions constructed and used to transiently cotransfect CHP-100 cells (Fig. might be involved in the interaction between GCS and the RTN 3B). We firstly confirmed the interaction between tagged GCS and proteins. RTN-1C in CHP-100 transfected cells by coimmunoprecipitation To verify whether the GCS-RTN interaction, highlighted by the (Fig. 3C) and subsequently analyzed their intracellular localization by two-hybrid method, takes place also in mammalian cells, we have confocal microscopy (Fig. 3D). Although RTN-1C is mainly detected carried out coimmunoprecipitation experiments of the native RTN-1C in the ER (green fluorescence), its localization is also detected on the and RTN-3 with GCS in CHP-100 cell extracts using an anti-GCS Golgi apparatus overlapping with the GCS (red fluorescence). The antibody. As shown in Fig. 3A, the Western blot analysis of the Golgi was identified by staining the cells with an anti-␤-CopI anti- immune complexes revealed that the endogenous RTN-1C coimmu- body (28). These findings support the above described interaction noprecipitated with GCS; by contrast, interaction between GCS and between GCS and RTN-1C by demonstrating that the two proteins RTN-3 could not be detected (data not shown). These results demon- colocalize in vivo in the Golgi apparatus or in regions of contact strated that although RTN-3 and RTN-1C share high homology, the (anchorage) between the ER and the Golgi itself (Fig. 3D). On the

Fig. 2. Identification of GCS-interactors. A, RTN-1C clones (four different clusters) identified by the two-hybrid screening; the first amino acid and the number of the clone selected are indicated. B, comparison of the primary structure of RTN-1C and RTN-3 gene products. The identical amino acids are highlighted in gray. The first amino acid of the clones selected in the two-hybrid screening and corresponding to four different clusters are indicated in red. C, diagram of the structure of RTN-1C and RTN-3. The first amino acids of the clones selected in the two-hybrid screening are indicated in red. D, model of the predicted structure for the RTN protein family.

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Fig. 3. Characterization of GCS/RTN-1C interaction. A, CHP-100 cell lysates were immunopre- cipitated with anti-GCS antibody or normal antirabbit antibody (NRS), used as a control, and were subjected to SDS-PAGE and Western blot using an anti-RTN-1C antibody. Samples were revealed also by anti-calnexin and anti-membrin antibodies. B, analysis of GFP-RTN-1C and RFP-GCS expression in CHP-100-transfected cells. C, cell lysates from CHP-100-transfected cells were immunopre- cipitated with anti-GCS antibody and were subjected to SDS-PAGE and Western blot using an anti-GFP antibody. D, CHP-100 cells were cotrans- fected with RFP-GCS and GFP-RTN-1C (top panels) and analyzed by confocal microscopy. The yellow staining in the overlaid images indicates the colocalization. Cells were transfected with RFP- GCS and immunostained with anti-␤-CopI antibody. The merge confirms the Golgi localization of GCS (bottom panels).

basis of these results, we hypothesized that the GCS/RTN-1C inter- RTN-1C effect on fenretinide is also suppressed by GCS inhibition action might play a role in the regulation of their respective functions. (Fig. 4, D and E), thus additionally indicating the involvement of To verify this hypothesis CHP-100 cells were transiently transfected GCS. Taken together, these results indicate that GCS/RTN-1C inter- with RTN-1C expression plasmid and the levels of RTN-1C overex- action may mediate signals between Golgi and ER compartments, pression analyzed by Western blot analysis (Fig. 4B). Transfected including the cellular response to apoptotic stimuli. In keeping with cells were then incubated with C6-NBD, a cell permeable synthetic these findings Tagami et al. (30) identified RTN-1C as a Bcl-xL- fluorescent substrate of GCS (29). In cells overexpressing RTN-1C, interactor and suggested a proapoptotic role for this protein. we detected a significant increase of GC levels compared with control Because apoptosis induction by ceramide requires its conversion to cells (Fig. 4A). By contrast, changes in the levels of sphingomyelin GSLs (31), the potentiation of fenretinide-induced apoptosis observed were not observed (data not shown), thus indicating that RTN-1C- in RTN-1C-transfected cells may be attributable to the GCS-depend- induced increase of GC is very likely because of its direct modulation ent conversion of preexisting ceramide into higher order GSLs or of GCS enzymatic activity. These results provide the first evidence for gangliosides. It has also been reported that ceramide levels increase in a GCS regulatory mechanism based on protein-protein interaction. response to fenretinide treatment, as a consequence of de novo syn-

Considering the involvement of GCS in feneretinide-induced apo- thesis; however, pretreatment of cells with fumonisin B1, an inhibitor ptosis previously demonstrated in this study, we next investigated the of ceramide synthase, does not affect fenretinide-induced cytotoxicity possibility that the GCS/RTN-1C interaction might play a role in the (16). In keeping with these findings, fenretinide-induced apoptosis retinoid-induced apoptosis. We analyzed whether the RTN-1C- may be dependent on ceramide metabolism, in particular on GSLs and dependent increase of GCS activity may affect fenretinide-induced gangliosides synthesis mediated by GCS. It is worth mentioning that apoptosis. CHP-100 cells were transiently transfected with RTN-1C in the GCS-antisense CHP-100 cells used in this study, the expression or RTN-3 expression plasmid (used as control) and treated with of gangliosides of higher order than GM3 is markedly reduced (14), fenretinide for 24 h. As shown in Fig. 4, C and D, we detected a thus supporting the notion of a strict correlation between GCS activ- specific increase of the apoptotic response to fenretinide in RTN-1C- ity, gangliosides biosynthesis, and cell death. It has been suggested transfected cells as compared with controls. Interestingly, the that GD3 is the major GSL candidate to mediate apoptosis; thus, it is 3863

Fig. 4. Effect of RTN-1C on fenretinide-induced apoptosis. A, CHP-100 cells, CHP-100-transfected with RTN-1C (CHP-RTN-1C), or vector control cells (CHP-GFP) were incubated Ϯ with NBD-C6 for 2 h, and GC levels were detected. Results are means SD of three independent determinations. B, Western blot analysis of RTN-1C overexpression in CHP-RTN-1C compared with CHP-GFP cells using an anti-RTN-1C antibody. C, CHP-100 transfected with vector control (CHP-GFP), RTN-1C (CHP-RTN-1C), or RTN-3 (CHP-RTN-3) were treated with 12 ␮M fenretinide (FenR) or ethanol control for 24 h, fixed, and stained with propidium iodide and analyzed by flow cytometry. D, quantitative analysis of data reported in Fig. 4C. Results are means Ϯ SD of three independent determinations. E, CHP-100 transfected with RTN-1C (CHP-RTN-1C) or vector control cells (CHP-GFP) were treated with 12 ␮M fenretinide (FenR) or ethanol control (ctr) for 24 h in the presence or absence of 20 ␮M PDMP, fixed, and stained with propidium iodide and analyzed by flow cytometry. F, .statistically significant (P Ͻ 0.05) compared with control cells ,ء .quantitative analysis of data reported in Fig. 4E. Results are means Ϯ SD of three independent determinations 3864

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. RTN-1C MODULATES GCS ACTIVITY IN CANCER CELLS likely that the increased apoptotic response to fenretinide observed in neuroblastoma by fenretinide or CD437 in combination with chemotherapeutic drugs. RTN-1C-transfected cells might be mediated by an increase of GD3 Int. J. Cancer, 88: 977–985, 2000. 16. Maurer, B. J., Melton, L., Billups, C., Cabot, M. C., and Reynolds, C. P. Synergistic levels (32, 33). cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and Finally, the data presented in this study demonstrate that GCS plays modulators of ceramide metabolism. J. Natl. Cancer Inst. (Bethesda), 92: 1897–1909, 2000. a key role in fenretinide-induced apoptosis, and this effect is influ- 17. Lovat, P. 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Federica Di Sano, Barbara Fazi, Gennaro Citro, et al.

Cancer Res 2003;63:3860-3865.

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