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[Cancer Research 65, 1887-1896, March 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Functional Proteomic Screen Identifies a Modulating Role for CD44 in Death Receptor–Mediated Apoptosis

Robert S. Hauptschein1, Kevin E. Sloan1, Claudia Torella2, Roya Moezzifard1, Maryann Giel-Moloney1, Carol Zehetmeier2, Christine Unger2, Leodevico L. Ilag2 and Daniel G. Jay1

1 Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts and 2 Xerion Pharmaceuticals, AG, Munich, Germany

Requests for reprints: Daniel G. Jay, Department of Physiology, Tufts University School of Medicine, 7th Floor, M&V Building, 136 Harrison Avenue, Boston, MA 02111. Phone: 617-636-6714; Fax: 617-636-0445; E-mail: daniel.jay{at}tufts.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand–mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand–mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.

Key Words: functional proteomics • apoptosis • CD44 • fluorophore-assisted light inactivation • death receptor


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Evading apoptosis is a fundamental pathophysiologic property of malignant cells that mediates their resistance to anticancer drugs (1). The elucidation of cellular apoptotic programs is an essential first step in the development of drugs targeting apoptosis resistance. Apoptotic signaling generally proceeds through the extrinsic and intrinsic pathways (2). The former transmits the apoptotic signal through death receptors (DR; e.g., Fas/CD95) upon binding to their cognate ligands [e.g., Fas ligand (FasL)], whereas the latter is centered on the mitochondria and the balance of specific mitochondria-associated proteins (e.g., BCL2, BAX) that respond to damage of cellular components. Initiation of either pathway leads to the activation of a proteolytic caspase cascade that mediates apoptosis (3).

Chemotherapeutic killing of tumor cells is largely mediated through apoptosis (4). Most apoptotic-promoting drugs in development for cancer therapy target the intrinsic pathway (5–7). Therefore, identifying novel cellular proteins that modulate the extrinsic pathway represents an alternative route to cancer therapeutics. Agonists to tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) DRs, which signal apoptosis selectively in tumor but not normal cells, have shown favorable results in inhibiting multiple cancers in animal studies (8–11). These treatments acted synergistically with standard chemotherapeutic drugs to cause enhanced antitumor activity (9). Whereas the systematic administration of FasL has severe toxicity on normal cells (12), modulation of Fas signaling could enhance tumor response to therapy. It has been shown that chemotherapeutic drug–induced apoptosis in many cells is mediated in part via Fas signaling, and the state of Fas signaling determines the efficacy of chemotherapeutic agents in some cancers (13). Identification of new apoptotic mediators will aid in the development of the next generation of target-specific chemotherapeutics.

Genomic and proteomic approaches to apoptosis constitute the most global means to identify all the genes and proteins involved in these pathways. Expression profiling comparisons between apoptotic and nonapoptotic cells by microarray or two-dimensional gel electrophoresis/mass spectrometry have netted many gene transcripts and proteins differing in abundance between these two conditions (14, 15). However, to show that these differentially expressed molecules are directly involved and functionally relevant to apoptosis, one must sequentially validate their function in a cellular system as a separate and time-costly step. This target validation of candidates is also a significant bottleneck in drug discovery (16, 17). To overcome this limitation, we and others have developed several complementary, human cell-based function-first proteomic strategies (16).

We have pioneered fluorophore-assisted light inactivation (FALI) as a novel tool for direct protein knockdown (18). FALI uses binders such as antibodies conjugated to a photosensitizing dye to focus light energy to inactivate or modify specific target proteins. We have previously established a functional proteomic screen using FALI to identify proteins important for tumor cell invasion (19, 20). Whereas this functional proteomic approach is thought to be generally applicable, the pairing of high-throughput screen (HTS)-FALI to other phenotypic readouts has not been directly tested. In this study, we have adapted FALI to screen multiple proteins in parallel and coupled it to a high-throughput caspase activity assay (HTS-FALIpop) to screen for proteins that regulate apoptosis. Using this system, we have identified CD44 as a modulator of DR-mediated apoptosis and propose a novel mechanism of regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture. HT-1080 fibrosarcoma, BT-549 mammary carcinoma, H9 T-cell lymphoma, and ACHN renal cell adenocarcinoma lines were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM (HT-1080, BT-549, ACHN) or RPMI 1640 (H9) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen, Carlsbad, CA; HyClone, Logan, UT). HT-1080 and ACHN were additionally supplemented with 0.1 mmol/L nonessential amino acids (Invitrogen). All cells were grown at 37°C under a humidified 5% CO2 atmosphere.

FITC Labeling of Antibodies. Antibodies were conjugated with FITC (pH 9.5; Molecular Probes, Eugene, OR) at room temperature as previously described (21). Fas antibodies included a receptor agonist CH-11, antagonist ZB4, and non-neutralizing UB2 monoclonal antibodies (Medical & Biological Laboratories, Woburn, MA). SFF-2 is a mouse monoclonal anti–pan CD44 antibody (Chemicon, Temecula, CA), and H-286 is a rabbit polyclonal anti-neuropilin-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). All antibodies recognize an extracellular epitope of their target protein. Control immunoglobulin G was obtained from Pierce (Rockford, IL).

FALI. Suspension cells, or adherent cells detached by Versene (Invitrogen) washing, were adjusted to 2.5 x 106 cells/mL in serum-free, phenol red–free HBSS (Invitrogen) and incubated with FITC-conjugated antibody for 1 hour at room temperature with gentle rocking. Cells were then transferred in at least sextuplet to replicate clear, flat-bottomed microtiter plates on ice. One plate was illuminated for 1 hour with 300 W (1 x 105 lux) blue-filtered light (Brilliant Blue no. 69, Roscolux, Stamford, CT), whereas the replicate control plate was kept in the dark for 1 hour.

Caspase Activity and Viability Assays. Following FALI, apoptosis was induced with either soluble human recombinant FLAG-tagged FasL, TNF{alpha}, or TRAIL and enhanced using an anti-FLAG cross-linking monoclonal antibody (Axxora, San Diego, CA) in serum-free, phenol red–containing DMEM or RPMI. The homogeneous caspases assay using quenched DEVD-rhodamine 110 substrate (Roche, Indianapolis, IN) and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium–based viability assay (Promega, Madison, WI) were done according to the manufacturer's recommendations. Fluorimetric or colorimetric measurements of the microtiter plates were read using a Tecan SpectraFluor Plus instrument (Research Triangle Park, NC).

Cell Surface Single-Chain Variable Fragment Antibody (scFv) Library Generation. Spleen RNA was harvested from HT-1080 immunized mice as described (19). cDNA was synthesized by PCR using a primer mix to amplify the immunoglobulin variable regions. Products were cloned into the phage display vector pXP10 and transfected into E. coli TG-1 resulting in a library size of 107 independent clones. HT-1080-specific scFvs were selected by immunopanning phage against fixed HT-1080, resulting in 2,760 binders. These scFvs were recloned into expression vector pXP7, containing His- and E-tags, and expressed in E. coli TG-1. scFv-containing bacterial lysates were prepared and additionally selected for binding to fixed HT-1080 cells by ELISA. These scFvs were confirmed to bind to the HT-1080 surface by immunocytochemistry on live cells. For HTS-FALIpop, the His-tagged scFvs were further purified from bacterial lysates by binding to Ni-NTA Superflow resin at pH 6.8 (Qiagen, Valencia, CA) and eluting at pH 4.5 in 300 mmol/L NaCl and 50 mmol/L NaH2PO4·H2O.

Immunoprecipitation and Mass Spectrometry. For scFv immunoprecipitations, the scFv gene was recloned into expression vector pXP14, containing a Strep-tag (19). Purified scFvs were coupled to StrepTactin Sepharose (50 µg/50 µL resin) and used to immunoprecipitate targets from HT-1080 lysate. The scFv-target complexes were eluted (10 mmol/L D-desthiobiotin and 0.1% Tween 20 in PBS), and captured proteins were analyzed by SDS-PAGE and silver staining. Stained bands were excised and trypsin digested. Resulting peptide fragments were extracted from the gel, desalted on ZipTip µC18, and spotted on a Teflon-coated matrix-assisted laser desorption/ionization target. The samples were analyzed using a STR-DE Voyager matrix-assisted laser desorption/ionization mass spectrometer (Applied Biosystems, Foster City, CA), and proteins were identified via peptide mass fingerprint, searching all entries for the species Homo sapiens in the National Center for Biotechnology Information and SwissProt databases.

Immunoblotting. Pelleted and PBS-washed cells were lysed and incubated in 0.2% NP40, 150 mmol/L NaCl, 20 mmol/L Tris (pH 7.5), and 10% glycerol with protease inhibitors (Roche) at 4°C. Following centrifugation and quantitation of the supernatant using DC Protein Assay (Bio-Rad, Hercules, CA), 50 µg of protein lysate was separated on a 4% to 15% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. Membranes were blocked in 5% nonfat dry milk, 0.05% Tween 20 in TBS, and reacted with mouse anti-caspase-8 (C15, Axxora), mouse anti-Fas (3D5, Axxora), mouse anti-Fas-associated death domain (A66-2, BD Biosciences, San Diego, CA), rabbit anti-poly(ADP-ribose) polymerase (Cell Signaling, Beverly, MA) and mouse anti-ß-actin and rabbit anti–pan actin antibodies (Sigma-Aldrich, St. Louis, MO). Primary antibodies were detected after enhanced chemiluminescence (Perkin-Elmer, Boston, MA) using peroxidase-conjugated anti-mouse (Cell Signaling) and anti-rabbit (Jackson ImmunoResearch, West Grove, PA) immunoglobulin G. The secondary antibodies were initially applied to fresh blots as controls. Optical densitometry of protein bands was determined using NIH Image software (http://rsb.info.nih.gov/nih-image/).

Death-Inducing Signaling Complex (DISC) Analysis. Cellular lysate, prepared as above, was incubated overnight at 4°C with rabbit anti-Fas polyclonal antibody (C20, Santa Cruz Biotechnology). Anti-rabbit immunoglobulin G agarose (Sigma-Aldrich) was added for 4 hours to capture the immune complexes. Beads were collected by centrifugation and washed four times in chilled lysis buffer. Immune complexes were eluted in PBS-sample loading buffer by boiling for 5 minutes before loading onto polyacrylamide gels.

CD44 Short Interfering RNA (siRNA). A double-stranded siRNA oligonucleotide targeting CD44 (5'-GAACGAAUCCUGAAGACAUdTdT-3') was chemically synthesized (Dharmacon, Lafayette, CO) and transfected into HT-1080 cells using Oligofectamine (Invitrogen) following manufacturer's instructions using 200 nmol/L siRNA per 10 cm dish. Cells were incubated with siRNA in OptiMEM (Invitrogen) for 6 hours after which time normal growth media was added. Cells were then incubated for 48 hours to achieve 90% knockdown of CD44 protein as measured by immunoblot. Control cells were transfected with a scrambled siRNA oligonucleotide at matching concentration. Cells were then challenged with FasL or vehicle control and assayed for caspase activation as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FALIpop Assay Detects Both Proapoptotic and Antiapoptotic Surface Molecules. It has previously been shown that FALI of Fas or caspase-3 can block apoptosis as measured by a viability assay (22). In this study, we sought to adapt FALI to a miniaturized format that would lend itself readily to high-throughput functional proteomic analysis (Fig. 1A). Cancer cells were distributed into 384-well microtiter plates and incubated with FITC-conjugated surface binders such that one binder could be interrogated per microwell. FALI was done by irradiating one plate with blue-filtered diffuse light, enabling multiple binders to be screened in parallel, whereas a replicate control plate was maintained in the dark. Following FALI, cells were challenged with an apoptotic inducer (or vehicle control). After a short incubation, cells were simultaneously lysed and incubated with DEVD-rhodamine 110, a profluorescent substrate of caspases (primarily executioner caspases-3/7; ref. 23). The amount of free, unquenched rhodamine 110 cleaved is proportional to caspase activity, a specific early/mid marker of apoptosis. Caspase activity was measured using an automated fluorescence plate reader. Binders that inactivate or modify a cellular protein involved in apoptosis were recognized as those that resulted in a significant increase or decrease in caspase activity in the presence of light. Immunoprecipitation and mass spectrometry with database searching were used to identify the cognate protein target of these binders.



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  		Figure 1. FALIpop assay allows high-throughput functional proteomic analysis. A, schematic overview of the assay. Surface binders conjugated to FITC were used to acutely inactivate their cellular protein targets by FALI. Cells were then challenged with an apoptotic inducer; following a short induction period, caspase activity was assayed. Binders that resulted in significant changes in caspase activity upon FALI (two rightmost wells) were then used to identify their protein targets. See text for further details. B, Fas-targeted FALI blocks FasL-mediated caspase activity induction. Fas-sensitive HT-1080 cells at 2.5 x 106 cells/mL were incubated with 6 µg/mL of the indicated FITC-conjugated antibody (Ab) for 1 hour and distributed at 105 cells/well in duplicate 96-well microtiter plates. The cell/antibody mixture was illuminated (+hV) or maintained in darkness (–hV). Immediately following FALI, cells were apoptotically induced with 160 ng/mL FasL and 1 µg/mL of cross-linking enhancer (+FasL) or kept noninduced (–FasL) for 4 hours before cells were lysed for caspase activity measurements. Columns, mean of triplicate samples; bars, SE. UB2, CH-11, and ZB4 are anti-Fas antibodies. Note that at the concentration of UB2 antibody used, it has both CH-11-like agonistic and ZB4-like antagonistic activity. FALI of Fas using UB2 abrogated FasL-mediated caspase induction (bracket). Results are representative of three independent experiments. *P < 0.01, Student's t test.

 
FALIpop was developed using FasL-induced apoptosis as this is a well-characterized apoptotic pathway with readily available reagents. FALI of Fas using the fluorescein-conjugated anti-Fas antibody (UB2) caused the specific loss of function of Fas, blocking FasL-mediated signaling in HT-1080 human fibrosarcoma cells (Fig. 1B). The reduction in caspase activity was large (over 80% inhibition), significant (P < 0.01), and antibody dose dependent (data not shown). There were no FALI-induced changes without antibody or with a species/isotype–matched nonimmune immunoglobulin control. Fas-targeted FALI also caused a 40% inhibition in baseline caspase activity. In addition, FALIpop can identify antibodies with direct functional effects on caspase activity in both baseline and FasL-stimulated apoptotic states. CH-11 is an agonistic antibody, and in this assay, resulted in a 9-fold increase in baseline caspase activity versus no antibody and nonimmune immunoglobulin G controls. FALI using the Fas function–blocking antibody ZB4 completely abrogated the normal 4-fold increase in caspase activity resulting from FasL treatment.

HTS-FALIpop Identifies Three scFv Targets that Modulate Apoptosis. We generated a surface-binding scFv library against HT-1080 cells (19). A phage display library was prepared from spleen mRNA derived from mice immunized with live and fixed HT-1080 cells. The expressed library of cloned immunoglobulin variable gene regions was affinity selected repeatedly against HT-1080. The resultant phage underwent ELISA to identify those that specifically bound to HT-1080 and then immunocytochemistry to confirm surface binding (data not shown). To establish proof of principle for a functional proteomic screen, 96 scFvs were screened by HTS-FALIpop (Fig. 2A). Criteria for positive scFvs were established as binders that resulted in large FALI-induced changes in FasL-mediated caspase induction that deviated beyond the average induction ± twice the average SD of all scFvs and that led to statistically significant changes (P < 0.001, Student's t test) compared with no light controls over multiple experiments. Whereas eight scFv targets caused large changes in caspase induction after FALI, we focused on the three (4a1, 4b2, and 4b3) with the highest statistical confidence level. FALI of all three of these targets resulted in consistent decreases in FasL-mediated caspase induction (24 ± 5%, 22 ± 3%, and 22 ± 7% inhibitions; mean ± SE). None of the scFvs exhibited a functional effect without FALI nor was there a significant effect on cellular baseline caspase activity (data not shown).



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  		Figure 2. HTS-FALIpop identifies scFv targets that modulate apoptosis. A, 96 scFvs to cell surface proteins were screened by HTS-FALIpop. FALI was done as described in Fig. 1 and Materials and Methods, except that scFvs were used at 20 µg/mL with HT-1080 cells. 2.5 x 104 antibody–treated or no antibody–treated cells were distributed to duplicate 384-well microtiter plates. Caspase induction is defined as the difference in caspase activity levels between the apoptotic-induced (+FasL) and the noninduced (–FasL) cell treatments. The results for the scFvs are ordered by magnitude of change in caspase induction from largest decrease to largest increase. The three positive-scoring scFvs (black) were those that caused both an induction change greater than the average induction of all scFvs ±2 x average SD (dashed lines) and were statistically significant within the highest confidence level (*P < 0.001) compared with no light controls. Gray bars, scFvs that had large FALI-mediated induction changes with P < 0.01; these targets were not pursued in this study. The positive control anti-Fas antibody (UB2) and negative control no antibody treatments were included in all experiments. Samples were assessed in triplicate and at least two independent experiments were done for each antibody. B, positive scFvs were confirmed by a cell viability assay. In these experiments, the apoptotic induction interval was extended up to 18 hours at which time the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay was done. A set of three scFvs (4a7, 4c4, and 5d3) that did not cause any detectable change in caspase activity served as matched-negative scFvs. Columns, averages of triplicate samples pooled from at least two independent experiments (normalized to the dark-treated control). *P < 0.05. For A and B: bars, SE.

 
We next confirmed these positive scFvs by a cellular viability assay, an end-stage apoptotic marker. Because the caspase activity assay records a snapshot of enzymatic activity, the decreases in caspase induction observed with these scFvs could be either due to early blockade of apoptosis induction or to accelerated cell death with diminution of enzyme activity by the time of measurement. By using a viability assay to measure the cumulative effect of FALI on cell survival, these two alternatives could be differentiated. Two positive scFvs (4a1 and 4b3), which caused a decrease in caspase activity upon FALI of their targets, showed a concurrent increase in cellular viability in the presence of FasL (38 ± 5% and 29 ± 8% increases; mean ± SE; Fig. 2B). These data establish that FALI of 4a1 and 4b3 is antiapoptotic. As the post-FALI interval for the viability assay is extended up to 18 hours (versus 4 hours for the caspase assay), this result also suggests that the protein turnover for these two targets in these cells is not rapid enough to allow for full compensation and restoration of FasL-mediated apoptosis due to de novo protein synthesis. In contrast, the effect of 4b2 may be partially masked by de novo protein synthesis.

The Positive scFv Target is CD44. The cognate antigen that is bound by positive scFv 4a1 was identified by immunoprecipitation and mass spectrometry. Immunoprecipitation using scFv 4a1 and HT-1080 cell lysate yielded a diffuse band at 80 to 85 kDa by silver staining (Fig. 3A). This band was excised, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization–mass spectrometry (Fig. 3B). The band was identified by peptide mass fingerprint database search as CD44 (SwissProt P16070, 81.5 kDa) with eight matching peptides covering 24% of the protein sequence (71 of 301 residues of the standard isoform; Fig. 3C). Nucleic acid sequencing revealed that positive scFv 4b3 was identical to 4a1 in its antigen-binding regions, suggesting that scFv 4b3 also targets CD44 (data not shown). The identification of CD44 was validated by performing FALI using an available monoclonal antibody specific to CD44 (Fig. 3D). CD44-targeted FALI using this antibody resulted in a large reduction in FasL-mediated caspase induction in the equivalent direction of the 4a1 scFv that was dose dependent and statistically significant.



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  		Figure 3. CD44 is the positive scFv target. A, immunoprecipitation using scFv 4a1 and HT-1080 lysate yields a dominant 80-85 kDa band (arrowhead). B, spectrum generated by matrix-assisted laser desorption/ionization analysis of tryptic digest of immunoprecipitated 80-85 kDa band. C, summary of peptide masses obtained in B. Eight of eight peptide fragments obtained matched SwissProt entry P16070 (CD44). D, CD44-targeted FALI inhibits FasL-mediated caspase induction. HT-1080 cells were treated with increasing concentrations (5, 10, 20, and 30 µg/mL) of FITC-conjugated scFv4a1 to CD44 and a commercially available pan CD44 monoclonal antibody (SFF-2). scFv 4a7 and an antibody to neuropilin-1 (expressed on HT-1080 cell surface; data not shown) served as negative controls and caused no significant change in caspase induction. Samples were assessed in triplicate. Columns, mean; bars, SE (*P < 0.01).

 
CD44-Targeted FALI Blocks DR-Mediated Apoptosis by Reducing DISC. We next examined whether FALI of CD44 protected against multiple ligands of the TNF DR family in HT-1080 cells. FALI-CD44 limited caspase induction by Fas, TRAIL, and TNF{alpha} by 90 ± 1%, 62 ± 1%, and 45 ± 0% (mean ± SE), respectively (Fig. 4A). Because these ligands utilize distinct receptors (24), these findings suggest that the antiapoptotic action of FALI-CD44 is not unique to Fas. To understand the mechanism of the apoptotic block by FALI-CD44, we tested whether the inhibition could be achieved in both Fas signaling type I and type II cells (25). One major distinction between these two types of cells is that type I cells do not depend on the mitochondrial pathway of Fas signaling for transmission of the apoptotic signal. We found that FALI-CD44 was antiapoptotic in both Fas type I (H9 and ACHN) and type II (HT-1080 and BT-549) cancer cells (Fig. 4B). Because CD44-targeted FALI inhibited apoptosis in Fas type I cells, it unlikely acts via the mitochondrial pathway of Fas signaling. Furthermore, because FALI-CD44 protected multiple human cancer cell lines of different tissue lineages from FasL-mediated apoptosis, the role of CD44 in DR-induced apoptosis may generally apply to multiple tumor types.



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  		Figure 4. A, FALI-CD44 reduces caspase induction by multiple ligands of the TNF{alpha} receptor family. Following FALI, apoptotically induced (+Ind) cells were incubated for 4 hours with 160 ng/mL FasL, 120 ng/mL TRAIL, or 480 ng/mL TNF{alpha}/10 µg/mL cycloheximide along with a cross-linking enhancer antibody at 1 µg/mL except for the TRAIL-challenged cells that received 2 µg/mL. Noninduced cell treatments (–Ind) were also included (*P < 0.0001). B, FALI-CD44 reduces FasL-mediated caspase induction in multiple human cancer cell lines, including Fas type I and II cells. Caspase activity was measured at 4 hours and 1 hour for the type II and I cells, respectively. All cell lines tested express CD44 (data not shown). For both A and B, samples were assessed in quadruplicate and are representative of two independent experiments. Columns, mean; bars, SE (*P < 0.01).

 
Apoptotic signaling through the extrinsic pathway commences with engagement of the DR by ligand resulting in receptor aggregation and formation of the DISC by recruitment of the adaptor protein Fas-associated death domain and procaspase-8 (26, 27). The juxtaposition of procaspase-8 molecules in the DISC is thought to result in their autoproteolysis and activation, which in turn cleaves procaspase-3, the major effector caspase (3). To determine the level of apoptotic block by FALI-CD44 more precisely, we tested the sensitivity of distinct steps of the Fas signaling pathway to FALI-CD44. CD44-targeted FALI reduced the accumulation of the activated caspase-8 cleavage products, p43/41 and p18, as well as the cleaved poly(ADP-ribose) polymerase product, a target of activated caspase-3, in the presence of FasL (Fig. 5A). Because FALI-CD44 inhibits both initiator and executioner caspase activation in HT-1080 (Fig. 5A), these data suggest that the apoptotic block lies upstream in Fas signaling.



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  		Figure 5. CD44-targeted FALI inhibits Fas DISC formation. A, FALI-CD44 blocks initiator and executioner caspase activation. Cell lysates from HT-1080 cells under conditions indicated were harvested 4 hours post-FALI. Fifty micrograms protein lysate were electrophoresed under reducing conditions and immunoblotted with the specified antibodies. The full-length (FL) proform of caspase-8 (55 kDa) is cleaved upon activation to products p43/41 and p18. B, FALI-CD44 reduces DISC assembly in Fas type I H9 cells (left). FITC-CD44 antibody–treated cells underwent the three designated treatments. Following a 30 minutes post-FALI interval, Fas was immunoprecipitated (IP) using equal amounts of lysate from 107 cells from the three different treatments. After capture, washing, and elution of the immune complexes, samples were subject to SDS-PAGE under reducing conditions and immunoblotted with the indicated antibodies. Cell lysates were also blotted (right) and probed with the specified antibodies. Bracket, FALI-CD44-induced ladder of Fas-containing high-molecular-weight species. FALI-CD44 decreased the amount of Fas-associated death domain coimmunoprecipitating in the Fas DISC and decreased the activation of procaspase-8 concomitant with the formation of a continuum of high-molecular-weight Fas-containing forms. Specificity was shown for these Fas-containing complexes by demonstrating their detection with two different anti-Fas-specific antibodies but not antibodies to other expressed surface proteins (data not shown). Actin detection was used as a loading control. The band that is seen in the Fas immunoblot in the IP-Fas samples (left) that is larger than the 48 kDa Fas represents the anti-Fas antibody used for the immunoprecipitation, as it is not present in the lysate-only samples (right). Results for A and B are representative of two independent experiments.

 
Because detection of the DISC is difficult in type II cells such as HT-1080 because of its low abundance (25), we analyzed DISC assembly in type I H9 cells. FALI-CD44 reduced the amount of Fas-associated death domain coimmunoprecipitating with Fas by ~40% in H9 (Fig. 5B). This decrease in DISC formation and caspase activation occurred concurrently with the generation of a pronounced ladder of Fas-containing high-molecular-weight species (Figs. 5B and 6). The reduced DISC assembly was not due to changes in Fas-associated death domain protein expression as these levels were unchanged by either FasL or FALI-CD44 treatment (Fig. 5B). FALI works by generating short-lived reactive singlet oxygen species that modify amino acid residues of a proximal protein target (18). Others have shown that reactive molecules are capable of cross-linking neighboring proteins (28). One can speculate that the Fas species observed at 48 kDa could be available to oligomerize in the DISC, whereas the FALI-CD44-shifted Fas species may be complexed and locked in a nonsignaling state, unavailable for recruitment into the DISC. We have also observed that one of the TRAIL receptors (DR5) was additionally present in a high-molecular-weight form following FALI-CD44 in HT-1080 cells (data not shown). The restoration of Fas apoptosis over time (Fig. 6) suggests the existence of a cellular recovery mechanism able to clear the nonfunctional Fas aggregates and replace them with intact Fas. This model suggests that Fas and other DRs are located in close proximity to CD44 in the plasma membrane where they are accessible to the short-lived singlet oxygen molecules and implicates CD44 in regulating DR availability.



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  		Figure 6. Time course of FALI-CD44-mediated protection from apoptosis induction. A, following CD44-targeted FALI, H9 cells were lysed after increasing intervals of FasL treatment to measure caspase activity. Columns, mean; bars, SE (*P < 0.01). B, cell lysates were collected from replicate wells to those in A (without FasL treatment) and subject to immunoblotting using the anti-Fas antibody (C20). Note that FALI-CD44-induced Fas-containing complexes are associated with apoptosis protection and decrease proportionally to the loss of protection from FALI-CD44 observed in A. Results for A and B are representative of two independent experiments.

 
To test whether CD44 might functionally regulate Fas availability, we used RNA interference (RNAi) to deplete the pool of CD44 in HT-1080 cells (Fig. 7A). CD44 knockdown cells were significantly more sensitive to FasL-induced caspase activation than scrambled controls (33 ± 3% increase in caspase activation; mean ± SE) in multiple, independent experiments (Fig. 7B). This result is consistent with a physiologic role for CD44 in sequestering Fas, making it less available for recruitment into the DISC complex (Fig. 7C). In summary, these data indicate that FALI-CD44 blocks apoptosis by inhibiting DISC formation and activation, likely by rendering DRs unavailable for DISC formation, and thus suggest a cellular role for CD44 in regulating DISC formation and sensitivity to apoptosis.



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  		Figure 7. A, CD44-specific siRNA duplexes were used to knock down protein expression by >90% by 48 hours. HT-1080 cells were transfected with siRNA targeting CD44 or with a scrambled control duplex. B, RNAi knockdown of CD44 in HT-1080 resulted in a significant increase in FasL-mediated caspase induction compared with control cells transfected with scrambled duplex. Columns, mean; bars, SE. *P < 0.01. Results for A and B are representative of three independent experiments. C, model of CD44 regulation of cell surface DR availability and DR-mediated apoptosis. We propose that DRs like Fas may be sequestered by CD44, making DR less available for DISC formation. Upon treatment with a death ligand (DL), such as FasL, DR is competed away from CD44 to form the DISC complex to initiate the apoptotic extrinsic pathway (left). CD44-targeted FALI generates a cloud of damaging reactive singlet oxygen species, which may cross-link neighboring surface DR molecules (middle, right). The cross-linked DRs would be unable to participate in the DISC rendering cells resistant to the death ligand. Depletion of CD44 molecules by RNAi would increase the number of free DR molecules available for death ligand binding and shift the apoptotic equilibrium to the left (bottom).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most comprehensive and direct means to identify all protein participants in apoptotic signaling is a functional proteomic approach. In this work, we have applied a novel functional proteomic strategy, FALI, to directly identify proteins that modulate FasL-mediated apoptosis. FALI targets light energy to cellular proteins in situ via a fluorophore, FITC, conjugated to a protein binder such as an antibody. The excited fluorophore generates damaging reactive singlet oxygen molecules that modify amino acid residues on a target protein, leading to structural alterations and functional inactivation or modulation of the protein (18). The short lifetime of these singlet oxygen species spatially restricts their damage to a 40 Å radius from the dye moiety, consistent with altering the targeted protein and potentially its nearest neighbors (18). As most antibodies are not function altering on their own, FALI is a proven method to generate function-modulating reagents with high probability (29). Indeed, FALI of Fas using the anti-Fas antibody UB2 abolishes almost all FasL-induced signaling (ref. 22; this study).

FALI is complementary to other functional proteomic and genomic approaches and offers several distinct advantages. Forward genetic screens using RNAi are powerful tools for gene knockdown, and several recent studies have proven productive (30, 31). Aza-Blanc et al. (30) used RNAi in a phenotypic screen to identify modulators of TRAIL-mediated apoptosis by targeting mostly known kinases. In contrast, we have done this study coupling FALI to FasL-induced apoptosis using an unbiased library of recombinant antibodies to surface proteins. We believe that this cellular compartment is the most technically accessible to antibodies and lends itself most readily to pharmacologic agents. FALI directly damages the cellular protein, allowing for immediate assessment of protein knockdown. The effects of FALI protein knockdown are acute, reducing the likelihood of cellular compensatory mechanisms and allowing for inactivation of proteins with slow turnover rates. Assuming a complete antibody repertoire, FALI has the capability to survey the functionality of all domains and forms of a multifunctional protein, distinguishing among isoforms due to alternative splicing and posttranslational events, such as phosphorylation and glycosylation. Thus, FALI can, in principle, interrogate the complete proteome. Furthermore, by not loading antibody into cells, it is possible to exclusively examine the function of the extracellular compartment of a targeted protein (19, 22).

We have generated thousands of surface protein–binding recombinant antibodies by phage display and immunopanning using HT-1080 fibrosarcoma cells. To show the feasibility of these reagents in a functional proteomic screen for apoptosis, a subset of scFvs were used with FALI. We coupled FALI to a facile and specific HTS of an apoptotic measure, namely caspase activation (HTS-FALIpop). Three of 96 scFv targets on HT-1080 cells resulted in large decreases in FasL-mediated caspase induction by HTS-FALIpop. One of these targets was identified as a known cell surface transmembrane protein, CD44. FALI using a previously characterized pan CD44–specific antibody confirmed this identification. CD44 has previously been implicated in apoptosis susceptibility (32–44) and thus establishes HTS-FALIpop as a valid functional proteomic approach to identify proteins that regulate apoptosis.

Unlike the well-characterized role of CD44, the predominant hyaluronan receptor, in enhancing tumor migration, invasion, and metastasis (45), there is no clear consensus on the direction and mechanisms in which CD44 regulates apoptosis. On the one hand, engagement of CD44 with its ligand hyaluronan or with CD44-specific antibodies has been shown to be proapoptotic in several cell types (32–35). T cells derived from CD44 null mice are more resistant than wild-type mice to concanavalin A–activated cell death (36). CD44 engagement up-regulates Fas and promotes Fas apoptosis in synovial cells (35). Conversely, CD44 engagement confers resistance to a variety of apoptotic inducers in several cancer cell lines (37–42). In some lung carcinoma cell lines, engagement of CD44 down-regulates surface Fas and Fas apoptosis (40). In other cell systems, CD44 engagement is associated with changes in the phosphoinositide 3-kinase/Akt cell survival pathway (39, 42). Confounding these mixed apoptotic outcomes that likely depend on the cellular context and the specific nature of the CD44 engagement (activation versus blockage) as well as the apoptotic inducer, CD44 is expressed as multiple isoforms due to alternative splicing and differential glycosylation and is also subject to complex regulation including phosphorylation and cleavage (45). For example, activation of a variant CD44 isoform but not the standard CD44 isoform in colon carcinoma cell lines caused apoptotic resistance to the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosurea (39). We sought to clarify the role of CD44 in DR-mediated apoptosis in our cell system.

As a first step to understand the mechanism through which CD44 may regulate DR signaling, we did CD44-targeted FALI and measured DISC formation, caspase-8 cleavage, and caspase-3 activation in Fas type I and II cells. DISC formation is relatively inefficient in Fas type II cells, and the execution of apoptosis requires amplification through a mitochondrial-based pathway (25). In these cells, small amounts of caspase-8 generated at the DISC truncate Bcl-2 interacting domain, which translocates to the mitochondria, resulting in cytochrome c release, apoptosome formation, and caspases-3/7 activation (2, 46). Therefore, inhibitors of the mitochondrial pathway block apoptosis in type II cells but have no impact on apoptosis in type I cells that form large amounts of DISC (25). We found that FALI-CD44 was able to block caspases-3/7 induction in both type I and II cells, indicating that modulation of CD44 does not exclusively affect the mitochondrial pathway. This result differs from previous findings (43, 44) that showed that CD44-deficient cells have decreased BCL-Xl levels and a shift of the apoptotic set point toward apoptosis. Our data suggest that CD44 regulation of DR-mediated apoptosis is not limited to the mitochondrial pathway. Also, in contrast to other cell systems (39, 42), we observed no changes in the levels of activated Akt or BAD following CD44-targeted FALI (data not shown).

FALI-CD44 was able to inhibit DISC formation, caspase-8 cleavage, and caspase-3 activation, demonstrating that the apoptotic block occurs at the most proximal event in DR signaling at the cell surface. We propose that Fas (and other DR) may be sequestered by CD44, making Fas unavailable for DISC formation (Fig. 7C). CD44 and Fas may be neighboring molecules on the cell surface such that CD44 damage by FALI changes the surface distribution of Fas, which makes it unavailable to form the DISC. The observation that Fas is present in a continuum of higher-order forms upon FALI treatment is consistent with this idea. These forms are stable after ß-mercaptoethanol treatment, suggesting that FALI of CD44 may covalently cross-link nearby Fas molecules. The ability of singlet oxygen species (the effectors of FALI) to cross-link neighboring proteins is well established (28). Similarly, treatment of cells with thiol-reactive cross-linkers has been shown to result in the inappropriate aggregation of Fas, preventing DISC assembly and activation (47). The nature of the proposed CD44 and Fas proximity is not known. Possibilities include a direct interaction, an association via a common-interacting molecule, or localization to a common cell membrane patch. We have been unable thus far to coimmunoprecipitate CD44 with Fas from cell lysates, possibly because their interaction may not be stable. RNAi-mediated knockdown of CD44 increased the sensitivity of HT-1080 cells to FasL-induced caspase activation. This result further supports the idea that CD44 may regulate the availability of Fas and thus sensitivity to DR-mediated apoptosis by sequestering Fas and making it less available for recruitment into an active DISC complex. Depletion of CD44 by RNAi would shift the apoptotic balance toward DISC formation, rendering cells more sensitive to FasL-induced apoptosis.

According to this model, up-regulation of CD44 generally observed in cancer (45) would thus reduce the availability of Fas and other DRs to activate apoptotic signaling and thereby enhance apoptosis resistance. This mechanism would be a novel basis of selection for apoptotic resistance underlying the association of elevated CD44 expression in many cancers. A precedent for this form of DR regulation as a cell survival mechanism in hepatocytes has recently been described (48). The MET growth factor receptor has been shown to sequester Fas in a MET/Fas complex, preventing Fas homo-oligomerization and FasL binding and thus rendering cells resistant to FasL-induced apoptosis. Evidence for a link between CD44 and Fas has been provided in vivo. Fas knockout mice develop a lymphoproliferative and autoimmune disease that is exacerbated when crossed with CD44 null mice (49). The combined effect of the double knockout is thought to result from increased protection of T cells from activation-induced cell death by apoptosis.

In summary, we have identified CD44 as a modulator of DR-mediated apoptosis in cancer cells through a novel functional proteomic strategy and have provided evidence that CD44 may elicit this regulation early in DR signaling at the level of DISC formation by controlling DR availability. These data also provide proof of concept for an expanded screen to the entire cancer proteome. In addition, this approach could readily be applied to identify modulators of resistance to alternative apoptotic programs, including chemotherapeutic-induced apoptosis.


   Acknowledgments
 
Grant support: National Cancer Institute grant CA-81668 and Goldhirsh Foundation (D.G. Jay), and NIH grant DK-07542 (R.S. Hauptschein, K.E. Sloan, and M. Giel-Moloney).

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 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Thomas Diefenbach, Julie Kerner, and Dean Yimlamai for helpful discussions and Claudina Aleman Stevenson, Brenda Eustace, and Jean Stewart for critical reading of the manuscript.


   Footnotes
 
Note: R.S. Hauptschein and K.E. Sloan contributed equally to this work.

Received 10/ 5/04. Revised 12/ 2/04. Accepted 12/21/04.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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