In this study we have explored the interaction between CD44 (the hyaluronic acid ( The transmembrane glycoprotein CD44 isoforms are all major hyaluronic acid (HA) 1 cell surface receptors that exist on many cell types, including macrophages, lymphocytes, fibroblasts, and epithelial cells (1-6). Because of their widespread occurrence and their role in signal transduction, CD44 isoforms have been implicated in the regulation of cell growth and activation as well as cell-cell and cell-extracellular matrix interactions (1-7). One of the distinct features of CD44 isoforms is the enormous heterogeneity in the molecular masses of these proteins. It is now known that all CD44 isoforms are encoded by a single gene that contains 19 exons (8). Of the 19 exons, 12 exons can be alternatively spliced (8). Most often, the alternative splicing occurs between exons 5 and 15, leading to an insertion in tandem of one or more variant exons (v1-v10 (exon 6-exon 14) in human cells) within the membrane-proximal region of the extracellular domain (8). The variable primary amino acid sequence of different CD44 isoforms is further modified by extensive N-and O-glycosylations and glycosaminoglycan additions (9 -12). In particular, CD44v3-containing isoforms have a heparin sulfate addition at the membrane-proximal extracellular domain of the molecule that confers the ability to bind heparin sulfate-binding growth factors (9, 10). Cell surface expression of CD44v isoforms changes profoundly during tumor metastasis, particularly during the progression of various carcinomas including breast carcinomas (13-17). In fact, CD44v isoform expression has been used as an indicator of metastasis.It has been shown that interaction between the cytoskeletal protein, ankyrin, and the cytoplasmic domain of CD44 isoforms plays an important role in CD44 isoform-mediated oncogenic signaling (6,18,19). Specifically, the ankyrin-binding domain (e.g. NGGNGTVEDRKPSEL between amino acids 306 and 320 in the mouse CD44 (20) and NSGNGAVEDRKPSGL amino acids 304 and 318 in human CD44 (21)) is required for the recruitment of Src kinase and the onset of tumor cell transformation (21). Furthermore, HA binding to CD44 stimulates a concomitant activation of p185 HER2 -linked tyrosine kinase (linked to CD44s via a disulfide linkage) and results in a direct cross-talk between two different signaling pathways (e.g. proliferation versus motility/invasion) (22). In tumor cells, the transmembrane linkage between CD44 isoform and the cytoskeleton promotes invasive and metastatic-specific tumor phenotypes (e.g. matrix degradation (matrix metalloproteinases) activities (23, 24), "invadopodia" formation (membrane projections), tumor cell invasion, and migration) (23). These findings strongly suggest that the interaction between CD44 isoform and the cytoskeleton plays a pivotal role in the onset of oncogenesis and tumor progression.The Rho family proteins (e.g. Rho, Rac, and Cdc42) are members of the Ras superfamily of GTP-binding proteins structurally related to but functionally dist...

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, ␣, ␤, ␥ forms but not the ␦ 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 downregulates oncogenic signaling events (e.g. Gab-1⅐PI 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-1⅐PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HAmediated breast cancer progression.

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 an in 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 HAdependent tumor cell migration, which may be required for human ovarian cancer progression.The cell adhesion molecule, CD44, is one of the major hyaluronic acid (HA) 1 receptors (1-3). It belongs to a family of transmembrane glycoproteins which contain a variable extracellular domain, a single spanning 23-amino acid transmembrane domain, and a 70-amino acid cytoplasmic domain (4). Nucleotide sequence analyses reveal that many CD44 isoforms (derived from alternative splicing mechanisms) are variants of the standard form, CD44s (4). CD44s (molecular mass ϳ85 kDa) is the most common isoform of CD44 found in many cell types including human ovarian carcinoma cells (5-9). The presence of high levels of CD44s (often together with CD44v) is emerging as an important metastatic tumor marker in a number of carcinomas, and is also implicated in the unfavorable prognosis of a variety of cancers including human ovarian cancers (5-9).The invasive phenotype of CD44s-positive epithelial tumor cells has been linked to HA-mediated CD44 signaling and cytoskeletal activation. CD44s contains several HA-binding sites in their extracellular domain (1-3). The binding of HA to CD44s causes cells to adhere to the extracellular matrix (ECM) components (1-3), and has also been implicated in the stimulation of several different biological activities (10 -16). The intracellular domain of CD44 binds to signaling proteins such as RhoGTPases (e.g. RhoA) (17); Tiam1, a guanine nucleotide exchange factor for Rac1 (18); and cytoskeletal proteins, including ankyrin (2,3,9,(17)(18)(19)(20)(21) and the ERM proteins (ezrin, radixin, and moesin) (23). Recent studies indicate that the binding of ECM components (e.g. HA) promote CD44-mediated Tiam1-Rac1 signaling and cytoskeleton function lea...

In this study we initially examined the interaction between CD44v3 (a hyaluronan (HA) receptor) and Vav2 (a guanine nucleotide exchange factor) in human ovarian tumor cells (SK-OV-3.ipl cell line). Immunological data indicate that both CD44v3 and Vav2 are expressed in SK-OV-3.ipl cells and that these two proteins are physically linked as a complex in vivo. By using recombinant fragments of Vav2 and in vitro binding assays, we have detected a specific binding interaction between the SH3-SH2-SH3 domain of Vav2 and the cytoplasmic domain of CD44. In addition, we have observed that the binding of HA to CD44v3 activates Vav2-mediated Rac1 signaling leading to ovarian tumor cell migration. Further analyses indicate that the adaptor molecule, growth factor receptor-bound protein 2 (Grb2) that is bound to p185 HER2 (an oncogene product), is also associated with the CD44v3-Vav2 complex. HA binding to SK-OV-3.ipl cells promotes recruitment of both Grb2 and p185 HER2 to the CD44v3-Vav2 complex leading to Ras activation and ovarian tumor cell growth. In order to determine the role of Grb2 in CD44v3 signaling, we have transfected SK-OV-3.ipl cells with Grb2 mutant cDNAs (e.g. ⌬N-Grb2 that has a deletion in the amino-terminal SH3 domain or ⌬C-Grb2 that has a deletion in the carboxyl-terminal SH3 domain). Our results clearly indicate that the SH3 domain deletion mutants of Grb2 (i.e. the ⌬N-Grb2 (and to a lesser extent the ⌬C-Grb2) mutant) not only block their association with p185 HER2 but also significantly impair their binding to the CD44v3-Vav2 complex and inhibit HA/CD44v3-induced ovarian tumor cell behaviors. Taken together, these findings strongly suggest that the interaction of CD44v3-Vav2 with Grb2-p185 HER2 plays an important role in the coactivation of both Rac1 and Ras signaling that is required for HA-mediated human ovarian tumor progression.The cell adhesion molecule, CD44, is a product of a single gene that undergoes alternative splicing of 12 possible exons to generate variant isoforms (1). Nucleotide sequence analyses reveal that the CD44 isoforms are variants of the standard form, CD44s (1). CD44s (molecular mass Ϸ85 kDa) is the most common isoform of CD44 found in many cell types including human ovarian carcinoma cells (2). CD44 can be further modified by extensive N-and O-glycosylations and glycosaminoglycan additions (3-5). Apparently, both post-translational modifications and/or alternative splicing within the CD44 structure determine the functional outcome of this molecule. CD44 is a transmembrane glycoprotein that is one of the major hyaluronan (HA) 1 receptors (6). CD44 binds to extracellular matrix components (i.e. HA) at its amino terminus of the extracellular domain (7,8). CD44 also contains specific binding sites for the cytoskeleton (9 -14) and various signaling molecules (15-19) within the 70-amino acid carboxyl terminus in the cytoplasmic domain.Both CD44 and HA appear to be overexpressed at sites of tumor attachment and are known to be involved in cell aggregation, proliferation, migration, and...

Purpose: Although tumor mutation burden (TMB) has been well known to predict the response to immune checkpoint inhibitors (ICI), lack of randomized clinical trial data has restricted its clinical application. This study aimed to explore the significance and feasibility of biomarker combination based on TMB and copy-number alteration (CNA) for the prognosis of each tumor and prediction for ICI therapy in metastatic pan-cancer milieu.Experimental Design: Non-ICI-treated MSK pan-cancer cohort was used for prognosis analysis. Three independent immunotherapy cohorts, including non-small cell lung cancer (n ¼ 240), skin cutaneous melanoma (n ¼ 174), and mixed cancer (Dana-Farber, n ¼ 98) patients from previous studies, were analyzed for efficacy of ICI therapy.Results: TMB and CNA showed optimized combination for the prognosis of most metastatic cancer types, and patients with TMB low CNA low showed better survival. In the predictive analysis, both TMB and CNA were independent predictive factors for ICI therapy. Remarkably, when TMB and CNA were jointly analyzed, those with TMB high CNA low showed favorable responses to ICI therapy. Meanwhile, TMB high CNA low as a new biomarker showed better prediction for ICI efficacy compared with either TMB-high or CNA-low alone. Furthermore, analysis of the non-ICI-treated MSK pan-cancer cohort supported that the joint stratification of TMB and CNA can be used to categorize tumors into distinct sensitivity to ICI therapy across pan-tumors.Conclusions: The combination of TMB and CNA can jointly stratify multiple metastatic tumors into groups with different prognosis and heterogeneous clinical responses to ICI treatment. Patients with TMB high CNA low cancer can be an optimal subgroup for ICI therapy.

In this study we have examined the interaction between CD44s (the standard form) and the p185 HER2 proto-oncogene in the ovarian carcinoma cell line. Surface biotinylation followed by wheat germ agglutinin column chromatography and anti-CD44-mediated immunoprecipitation indicate that both CD44s and p185 HER2 are expressed on the cell surface and most importantly, that these two molecules are physically linked to each other via interchain disulfide bonds. We have also determined that hyaluronic acid stimulates CD44s-associated p185 HER2 tyrosine kinase activity, leading to an increase in the ovarian carcinoma cell growth.After transfection of the ovarian carcinoma cell line with the adenovirus 5 E1A gene, which is known to repress p185 HER2 expression, we observed that both surface CD44s expression and CD44s-mediated cell adhesion to hyaluronic acid are significantly reduced in the transfectant cells compared with the control cells. These data suggest that down-regulation of p185 HER2 blocks CD44s expression and subsequent adhesion function. Our findings also indicate that the CD44s-p185 HER2 interaction is both functionally coupled and biosynthetically regulated. We believe that direct "cross-talk" between these two surface molecules (i.e. CD44s and the p185 HER2 ) may be one of the most important signaling events in human ovarian carcinoma development.

In this study we have examined the interaction between CD44 (a hyaluronan (HA) receptor) and the trans-

Metastatic breast tumor Met‐1 cells express CD44v3,8–10, a major adhesion receptor that binds extracellular matrix components at its extracellular domain and interacts with the cytoskeletal protein, ankyrin, at its cytoplasmic domain. In this study, we have determined that CD44v3,8–10 and RhoA GTPases are physically associated in vivo, and that CD44v3,8–10‐bound RhoA displays GTPase activity, which can be inhibited by botulinum toxin C3‐mediated ADP‐ribosylation. In addition, we have identified a 160 kDa Rho‐Kinase (ROK) as one of the downstream targets for CD44v3,8–10‐bound RhoA GTPase. Specifically, RhoA (complexed with CD44v3,8–10) stimulates ROK‐mediated phosphorylation of certain cellular proteins including the cytoplasmic domain of CD44v3,8–10. Most importantly, phosphorylation of CD44v3,8–10 by ROK enhances its interaction with the cytoskeletal protein, ankyrin. We have also constructed two ROK cDNA constructs that encode for proteins consisting of 537 amino acids [designated as the constitutively active form of ROK containing the catalytic domain (CAT, also the kinase domain)], and 173 amino acids [designated as the dominant‐negative form of ROK containing the Rho‐binding domain (RB)]. Microinjection of the ROK's CAT domain into Met‐1 cells promotes CD44‐ankyrin associated membrane ruffling and projections. This membrane motility can be blocked by CD44 antibodies and cytochalasin D (a microfilament inhibitor). Furthermore, overexpression of a dominant‐negative form of ROK by transfection of Met‐1 cells with ROK's Rho‐binding (RB) domain cDNA effectively inhibits CD44‐ankyrin‐mediated metastatic behavior (e.g., membrane motility and tumor cell migration). These findings support the hypothesis that ROK plays a pivotal role in CD44v3,8–10‐ankyrin interaction and RhoA‐mediated oncogenic signaling required for membrane‐cytoskeleton function and metastatic tumor cell migration. Cell Motil. Cytoskeleton 43:269–287, 1999. © 1999 Wiley‐Liss, Inc.