ABSTRACT
In the earliest stages of metastasis, breast cancer cells must reorganize the cytoskeleton to affect cell shape change and promote cell invasion and motility. These events require the cytoskeletal regulators Cdc42 and Rho, their effectors such as N-WASp/WAVE, and direct inducers of actin polymerization such as Arp2/3. Little consideration has been given to molecules that shape the cell membrane. The F-BAR proteins CIP4, TOCA-1, and FBP17 generate membrane curvature and act as scaffolding proteins for activated Cdc42 and N-WASp. We found that expression of CIP4, but not TOCA-1 or FBP17, was increased in invasive breast cancer cell lines in comparison with weakly or noninvasive breast cancer cell lines. Endogenous CIP4 localized to the leading edge of migrating cells and to invadopodia in cells invading gelatin. Because CIP4 serves as a scaffolding protein for Cdc42, Src, and N-WASp, we tested whether loss of CIP4 could result in decreased N-WASp function. Interaction between CIP4 and N-WASp was epidermal growth factor responsive, and CIP4 silencing by small interfering RNA caused decreased tyrosine phosphorylation of N-WASp at a Src-dependent activation site (Y256). CIP4 silencing also impaired the migration and invasion of MDA-MB-231 cells and was associated with decreased formation of invadopodia and gelatin degradation. This study presents a new role for CIP4 in the promotion of migration and invasion of MDA-MB-231 breast cancer cells and establishes the contribution of F-BAR proteins to cancer cell motility and invasion.
Subject(s)
Breast Neoplasms/pathology , Cell Surface Extensions/pathology , Microtubule-Associated Proteins/physiology , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Animals , Blotting, Western , Breast Neoplasms/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Adhesion , Cell Movement , Cell Proliferation , Cells, Cultured , Fatty Acid-Binding Proteins , Female , Fluorescence Resonance Energy Transfer , Fluorescent Antibody Technique , Gelatin/metabolism , Humans , Immunoenzyme Techniques , Immunoprecipitation , Mice , Mice, Nude , Minor Histocompatibility Antigens , Neoplasm Invasiveness , Phosphorylation , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tyrosine/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/genetics , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Estrogen receptor alpha (ER alpha) is a ligand-activated transcription factor that regulates expression of estrogen-responsive genes. Upon binding of the ligand-occupied ER alpha to estrogen response elements (EREs) in DNA, the receptor interacts with a variety of coregulatory proteins to modulate transcription of target genes. We have isolated and identified a number of proteins associated with the DNA-bound ER alpha. One of these proteins, Rho guanosine diphosphate (GDP) dissociation inhibitor alpha (RhoGDI alpha), is a negative regulator of the Rho family of GTP-binding proteins. In this study, we demonstrate that endogenously expressed RhoGDI alpha is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, and that RhoGDI alpha binds directly to ER alpha, alters the ER alpha-ERE interaction, and influences the ability of ER alpha to regulate transcription of a heterologous estrogen-responsive reporter plasmid in transient transfection assays as well as endogenous, estrogen-responsive genes in MCF-7 cells. Our studies suggest that, in addition to the activity of RhoGDI alpha in the cytoplasm, it also influences ER alpha signaling in the nucleus.
