Functional proteomic screen identifies a modulating role for CD44 in death receptor-mediated apoptosis

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 s...

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Bibliographic Details
Published in:	Cancer research (Chicago, Ill.) Vol. 65; no. 5; pp. 1887 - 1896
Main Authors:	HAUPTSCHEIN, Robert S, SLOAN, Kevin E, TORELLA, Claudia, MOEZZIFARD, Roya, GIEL-MOLONEY, Maryann, ZEHETMEIER, Carol, UNGER, Christine, ILAG, Leodevico L, JAY, Daniel G
Format:	Journal Article
Language:	English
Published:	Philadelphia, PA American Association for Cancer Research 01-03-2005
Subjects:	Animals Antineoplastic agents Apoptosis Biological and medical sciences Carcinoma, Renal Cell - metabolism Carcinoma, Renal Cell - pathology Caspases - metabolism Death Domain Receptor Signaling Adaptor Proteins Enzyme Activation fas Receptor - genetics fas Receptor - metabolism Fibrosarcoma - metabolism Fibrosarcoma - pathology Humans Hyaluronan Receptors - genetics Hyaluronan Receptors - metabolism Immunoglobulin Fragments - immunology Kidney Neoplasms - metabolism Kidney Neoplasms - pathology Lymphoma, T-Cell - metabolism Lymphoma, T-Cell - pathology Mammary Neoplasms, Experimental - metabolism Mammary Neoplasms, Experimental - pathology Mass Spectrometry Medical sciences Mice Pharmacology. Drug treatments Proteomics Receptors, Tumor Necrosis Factor - metabolism Tumor Cells, Cultured Functional Cell death Mortality Proteomics Medical screening Apoptosis Biological receptor
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0008-5472 1538-7445
DOI:	10.1158/0008-5472.can-04-3571