Lilly Y. W. Bourguignon

Nuclear localization of EGF receptor and its potential new role as a transcription factor

Epidermal growth factor receptor (EGFR) has been detected in the nucleus in many tissues and cell lines. However, the potential functions of nuclear EGFR have largely been overlooked. Here we demonstrate that nuclear EGFR is strongly correlated with highly proliferating activities of tissues. When EGFR was fused to the GAL4 DNA-binding domain, we found that the carboxy terminus of EGFR contained a strong transactivation domain. Moreover, the receptor complex bound and activated AT-rich consensus-sequence-dependent transcription, including the consensus site in cyclin D1 promoter. By using chromatin immunoprecipitation assays, we further demonstrated that nuclear EGFR associated with promoter region of cyclin D1 in vivo. EGFR might therefore function as a transcription factor to activate genes required for highly proliferating activities.

Signaling Properties of Hyaluronan Receptors

In 1979, hyaluronan was demonstrated to bind specifically and with high affinity to intact cells (1), and in 1980, it was shown to enhance cell motility on two-dimensional culture surfaces where the hydrodynamic properties of hyaluronan were not necessary to open spaces for cells to move into (2). These two demonstrations raised the possibility that hyaluronan had the potential to directly modify cell behavior. In 1989, hyaluronan was shown to promote protein tyrosine phosphorylation cascades (3) that were later proven to be required for hyaluronan-mediated motility on planar culture surfaces (4). Since then, small amounts (nanograms) of hyaluronan have been shown to activate a variety of protein tyrosine and serine/threonine kinases. These include the non-receptor protein tyrosine kinase Src (5, 6), HER2/Neu receptor (7), focal adhesion kinase (4, 8–10), protein kinase C (11, 12), and MAP kinases (9, 10). Likely as a consequence of regulating these kinases, hyaluronan promotes expression of specific cytokines and proteins involved in extracellular matrix remodeling (e.g. Ref. 13). The study of murine cardiac cells derived from hyaluronan synthase 2 (HAS2) knockout mice has provided the most convincing evidence for a signaling capability of hyaluronan (14). HAS2 / cardiac cells do not undergo an endothelial-mesenchymal transformation associated with migration from tissue explants whereas wild-type cells do (14). However, the addition of nanogram amounts of exogenous hyaluronan “rescues” knockout cells. Furthermore, a dominant negative mutant of the small GTPase, Ras, blocks the effects of exogenous hyaluronan (14). These results suggest that hyaluronan signals through Ras to regulate motility and are consistent with previous studies showing that exogenous hyaluronanreceptor interactions regulate Ras signaling (4, 8). This ability of hyaluronan to activate intracellular signaling cascades requires interactions with cell-associated hyaluronan-binding proteins or hyaladherins (15) but is additionally modified by the amount and size of hyaluronan present in the environment of the cell. Further, not all cell types activate signaling cascades in response to hyaluronan (11), indicating that cell background is also an important determinant. Here, we review current understanding of the mechanisms by which hyaluronan signals.

CD44 Interaction with Tiam1 Promotes Rac1 Signaling and Hyaluronic Acid-mediated Breast Tumor Cell Migration

In this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line). Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo. Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e. the NH2-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393–738 of Tiam1) and an in vitrobinding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K d ) of 0.2 nm, which is comparable with CD44 binding (K d = ∼0.13 nm) to intact Tiam1. These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44. Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration. Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration. Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors. Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression.

Hyaluronan-CD44 Interaction Activates Stem Cell Marker Nanog, Stat-3-mediated MDR1 Gene Expression, and Ankyrin-regulated Multidrug Efflux in Breast and Ovarian Tumor Cells

Hyaluronan (HA) is a major glycosaminoglycan in the extracellular matrix whose expression is tightly linked to multidrug resistance and tumor progression. In this study we investigated HA-induced interaction between CD44 (a HA receptor) and Nanog (an embryonic stem cell transcription factor) in both human breast tumor cells (MCF-7 cells) and human ovarian tumor cells (SK-OV-3.ipl cells). Using a specific primer pair to amplify Nanog by reverse transcriptase-PCR, we detected the expression of Nanog transcript in both tumor cell lines. In addition, our results reveal that HA binding to these tumor cells promotes Nanog protein association with CD44 followed by Nanog activation and the expression of pluripotent stem cell regulators (e.g. Rex1 and Sox2). Nanog also forms a complex with the “signal transducer and activator of transcription protein 3” (Stat-3) in the nucleus leading to Stat-3-specific transcriptional activation and multidrug transporter, MDR1 (P-glycoprotein) gene expression. Furthermore, we observed that HA-CD44 interaction induces ankyrin (a cytoskeletal protein) binding to MDR1 resulting in the efflux of chemotherapeutic drugs (e.g. doxorubicin and paclitaxel (Taxol)) and chemoresistance in these tumor cells. Overexpression of Nanog by transfecting tumor cells with Nanog cDNA stimulates Stat-3 transcriptional activation, MDR1 overexpression, and multidrug resistance. Down regulation of Nanog signaling or ankyrin function (by transfecting tumor cells with Nanog small interfering RNA or ankyrin repeat domain cDNA) not only blocks HA/CD44-mediated tumor cell behaviors but also enhances chemosensitivity. Taken together, these findings suggest that targeting HA/CD44-mediated Nanog-Stat-3 signaling pathways and ankyrin/cytoskeleton function may represent a novel approach to overcome chemotherapy resistance in some breast and ovarian tumor cells displaying stem cell marker properties during tumor progression.

CD44v3,8–10 is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells

In the present study, we have employed a unique breast cancer cell line (Met‐1, which was derived from a high metastatic potential tumor in transgenic mice expressing polyomavirus middle T oncogene) to study the role of CD44 variant isoform(s) in the regulation of metastatic breast tumor cell behavior. The results of reverse transcriptase–polymerase chain reaction, Southern blot, nucleotide sequencing, immunoprecipitation, and immunoblot analyses indicated that these cells express a major CD44 isoform (molecular weight ≈260 kDa) containing a v3,8–10 exon insertion (designated as CD44v3,8–10). In addition, we have determined that CD44v3,8–10 binds specifically to the cytoskeletal proteins such as ankyrin. Biochemical analyses, using competition binding assays and a synthetic peptide identical to NGGNGTVEDRKPSEL (a sequence located between aa480 and aa494 of CD44v3,8–10) indicate that this 15‐amino acid peptide binds specifically to the cytoskeletal protein ankyrin (but not to fodrin or spectrin). This peptide competes effectively for ankyrin binding to CD44v3,8–10. Therefore, we believe that the sequence 480NGGNGTVEDRKPSE494L, located at the cytoplasmic domain of CD44v3,8–10, is required for the ankyrin binding. We have also detected that CD44v3,8–10‐containing Met‐1 cells are capable of forming membrane spikes or “invadopodia” structures and undergo active migration processes. Treatments of Met‐1 cells with certain agents including anti‐CD44v3 antibody, cytochalasin D (a microfilament inhibitor), and W‐7 (a calmodulin antagonist), but not colchicine (a microtubule disrupting agent) effectively inhibit “invadopodia” formation and subsequent tumor cell migration. Further analyses using zymography assays and double immunofluorescence staining indicated that CD44v3,8–10 is closely associated with the active form of matrix metalloproteinase, MMP‐9, in a complex within “invadopodia” structures. These findings suggest that CD44v3,8–10 plays an important role in linking ankyrin to the membrane‐associated actomyosin contractile system required for “invadopodia” formation (coupled with matrix degradation activities) and tumor cell migration during breast cancer progression. J. Cell. Physiol. 176:206–215, 1998.

Hyaluronan-CD44 Interaction with Protein Kinase Cϵ Promotes Oncogenic Signaling by the Stem Cell Marker Nanog and the Production of MicroRNA-21, Leading to Down-regulation of the Tumor Suppressor Protein PDCD4, Anti-apoptosis, and Chemotherapy Resistance in Breast Tumor Cells

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cϵ (PKCϵ), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCϵ activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCϵ or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCϵ-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCϵ-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.

CD44 Interaction with c-Src Kinase Promotes Cortactin-mediated Cytoskeleton Function and Hyaluronic Acid-dependent Ovarian Tumor Cell Migration

In this study we have demonstrated that both CD44 (the hyaluronan (HA) receptor) and c-Src kinase are expressed in human ovarian tumor cells (SK-OV-3.ipl cell line), and that these two proteins are physically associated as a complex in vivo. Using a recombinant cytoplasmic domain of CD44 and anin vitro binding assay, we have detected a specific interaction between CD44 and c-Src kinase. Furthermore, the binding of HA to SK-OV-3.ipl cells promotes c-Src kinase recruitment to CD44 and stimulates c-Src kinase activity, which, in turn, increases tyrosine phosphorylation of the cytoskeletal protein, cortactin. Subsequently, tyrosine phosphorylation of cortactin attenuates its ability to cross-link filamentous actin in vitro. In addition, transfection of SK-OV-3.ipl cells with a dominant active form of c-Src (Y527F)cDNA promotes CD44 and c-Src association with cortactin in membrane projections, and stimulates HA-dependent/CD44-specific ovarian tumor cell migration. Finally, overexpression of a dominant-negative mutant of c-Src kinase (K295R) in SK-OV-3.ipl cells impairs the tumor cell-specific phenotype. Taken together, these findings strongly suggest that CD44 interaction with c-Src kinase plays a pivotal role in initiating cortactin-regulated cytoskeleton function and HA-dependent tumor cell migration, which may be required for human ovarian cancer progression.

Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression.

Hyaluronan (HA), a major component of the extracellular matrix (ECM), is enriched in many types of tumors. In cancer patients HA concentrations are usually higher in malignant tumors than in corresponding benign or normal tissues, and in some tumor types the level of HA is predictive of malignancy. HA is often bound to CD44 isoforms which are ubiquitous, abundant, and functionally important cell surface receptors. This article reviews the current evidence for HA/CD44-mediated activation of the ankyrin-based cytoskeleton and RhoGTPase signaling during tumor progression. A special focus is placed on the role of HA-mediated CD44 interaction with unique downstream effectors (e.g., the cytoskeletal protein, ankyrin and/or various GTPases (e.g., RhoA, Rac1 and Cdc42)) in coordinating intracellular signaling pathways (e.g., Ca(2+) mobilization, Rho signaling, PI3 kinase-AKT activation, NHE1-mediated cellular acidification, transcriptional upregulation and cytoskeletal function) and generating the concomitant onset of tumor cell activities (e.g., tumor cell adhesion, growth, survival, migration and invasion) and tumor progression. I believe this information will provide valuable new insights into poorly understood aspects of solid tumor malignancy. Furthermore, the new knowledge concerning HA/CD44-mediated oncogenic signaling events will have potentially important clinical utility, and could establish CD44 and its associated signaling molecules as important tumor markers for the early detection and evaluation of oncogenic potential. It could also serve as ground work for the future development of new drug targets to inhibit HA/CD44-mediated tumor metastasis and cancer progression.

Capping and the Cytoskeleton

Publisher Summary This chapter summarizes the results obtained in capping/cytoskeleton research. There are two types of capping phenomena: ligand-dependent and ligand-independent processes. A number of different reagents, such as colchicine and hypertonic media, can cause the formation of cap structures in the absence of any externally added ligand. Morphological studies, using both transmission and scanning electron microscopy, of cells forming caps in hypertonic medium have identified a strong association between cap structures and microvilli. External ligand (antibody, lectin, or some hormones) mediated receptor patching and capping is responsible for important immune-related responses: ( 1) proliferation and differentiation of the cells into antibody-secreting plasma cells, (2) the development of an increased number of precursor cells which are responsible for immunological memory, and (3) the activation and proliferation of T cells which carry out cell-mediated cytotoxic killing. In nonimmune cells, capping may also be involved in important cellular processes, such as endocytosis, chemotaxis, mitogenesis, and general cell–cell recognition.

Hyaluronan-CD44 interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/Ras co-activation, phospholipase C epsilon-Ca2+ signaling, and cytoskeleton modification in head and neck squamous cell carcinoma cells.

In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase Cϵ (PLCϵ) activity. In particular, the activation of RhoA-PLCϵ by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLCϵ-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLCϵ-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.