Tetraspanin proteins CD9 and CD81 regulate sperm-egg fusion, cell growth and proliferation, tumor cell motility and metastasis, and other functions. CD9 and CD81 do not serve in a typical cell surface receptor-ligand binding capacity. Rather they function in the context of multicomponent proteolipid complexes known as Tetraspanin-Enriched Microdomains (TEMs). Within TEMs, CD9 and CD81 each can directly associate with Ig superfamily transmembrane proteins known as EWI-2 and EWI-F (named for conserved Glu-Trp-Ile motifs). We suggest that EWI-2 and EWI-F are major participants in many of the functions previously ascribed to CD9 and CD81. Although sharing many of the same components, CD9/CD81-EWI-2 and CD9/CD81-EWI-F complexes differ in size and density. Furthermore, CD9/CD81-EWI-2 complexes depress cell spreading, invasion and migration;are missing from many tumor cells;impair intracranial glioblastoma growth;and are associated with improved glioblastoma patient prognosis. Conversely, CD9/CD81-EWI-F complexes are upregulated on many tumor cells, enhance cell survival, and are associated with poor glioblastoma patient prognosis. Our central guiding hypothesis is that EWI-2 and EWI-F complexes function in opposition on tumor cells. Hence, our major goals regarding EWI-2 and EWI-F complexes are to i) characterize more fully their opposing cell biological functions, ii) understand their functions in the context of TEMs, and iii) examine opposing effects on tumor cells in vivo, using a human glioblastoma/nude mouse xenograft model. More specifically, in Aim 1 we will examine EWI-2 and EWI-F effects on cell motility, morphology, invasion, adhesion, and survival;and we will examine their subcellular localization.
In Aim 2, we will disrupt protein palmitoylation, which plays an essential role in holding together TEMs. This will be done by removing or inhibiting the protein acyltransferase(s) needed for tetraspanin and EWI protein palmitoylation, and by mutating specific EWI-2 and EWI-F palmitoylation sites.
In Aim 3, we will use a quantitative proteomics approach to identify specific protein partners that preferentially associate with EWI-2 or EWI-F. Such proteins may hold the key towards understanding their opposing functions.
In Aim 4, we will investigate the contributions of EWI-2 and EWI-F cytoplasmic domains towards their functions, with a focus on key signaling pathways (involving PKC and AKT), and on specific molecular interactions. Finally, in Aim 5 we will express wild type and mutant forms of EWI-2 and EWI-F in glioblastoma cells, in order to determine effects on intracranial tumor growth. Public Health Relevance: EWI-2 and EWI-F protein complexes have opposing functions - the former retards tumor progression, while the latter enhances it. By understanding the molecular details regarding these newly discovered molecules, we will gain new insights and new tools that can be used to combat cancer. Findings will be relevant not only to brain cancer (studied here), but also for other types of cancer, as well as for normal cell functions that involve EWI-2 and EWI-F.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038903-25
Application #
8136455
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Flicker, Paula F
Project Start
1987-08-01
Project End
2012-08-31
Budget Start
2011-09-01
Budget End
2012-08-31
Support Year
25
Fiscal Year
2011
Total Cost
$317,033
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
076580745
City
Boston
State
MA
Country
United States
Zip Code
02215
Malhotra, Deepali; Fletcher, Anne L; Astarita, Jillian et al. (2012) Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks. Nat Immunol 13:499-510
Sharma, Chandan; Rabinovitz, Isaac; Hemler, Martin E (2012) Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin ?6?4. Cell Mol Life Sci 69:2233-44
Wang, Hong-Xing; Li, Qinglin; Sharma, Chandan et al. (2011) Tetraspanin protein contributions to cancer. Biochem Soc Trans 39:547-52
Wang, Hong-Xing; Kolesnikova, Tatiana V; Denison, Carilee et al. (2011) The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization. J Cell Sci 124:2702-10
Lafleur, Marc A; Xu, Daosong; Hemler, Martin E (2009) Tetraspanin proteins regulate membrane type-1 matrix metalloproteinase-dependent pericellular proteolysis. Mol Biol Cell 20:2030-40
Xu, Daosong; Sharma, Chandan; Hemler, Martin E (2009) Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein. FASEB J 23:3674-81
Kolesnikova, Tatiana V; Kazarov, Alexander R; Lemieux, Madeleine E et al. (2009) Glioblastoma inhibition by cell surface immunoglobulin protein EWI-2, in vitro and in vivo. Neoplasia 11:77-86, 4p following 86
Sharma, Chandan; Yang, Xiuwei H; Hemler, Martin E (2008) DHHC2 affects palmitoylation, stability, and functions of tetraspanins CD9 and CD151. Mol Biol Cell 19:3415-25
Hemler, Martin E (2008) Targeting of tetraspanin proteins--potential benefits and strategies. Nat Rev Drug Discov 7:747-58
Fiorentino, Michelangelo; Zadra, Giorgia; Palescandolo, Emanuele et al. (2008) Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of beta-catenin in prostate cancer. Lab Invest 88:1340-8

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