Hyaluronan-mediated CD44 interaction with RhoGEF and Rho kinase promotes Grb2-associated binder-1 phosphorylation and phosphatidylinositol 3-kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression.
In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, alpha, beta, gamma forms but not the delta form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1.PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1.PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression.
Bourguignon
Lilly Y W
LY
Department of Medicine, University of California at San Francisco and the Endocrine Unit (111N), Veterans Affairs Medical Center, San Francisco, Calfornia 94121, USA. lillyb@itsa.ucsf.edu
Singleton
Patrick A
PA
Zhu
Hongbo
H
Diedrich
Falko
F
D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000378:metabolism; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology; D019556:COS Cells; D002455:Cell Division; D002470:Cell Survival; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D018450:Disease Progression; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000494:pharmacology*; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000378:metabolism; D008954:Models, Biological; D009361:Neoplasm Invasiveness; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D054460:rho-Associated Kinases
D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate; D000276:Adjuvants, Immunologic; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D051380:GRB2 Adaptor Protein; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D016173:Macrophage Colony-Stimulating Factor; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D054460:rho-Associated Kinases; C098587:GAB1 protein, human; C495948:GRB2 protein, human