Stem cell marker (Nanog) and Stat-3 signaling promote MicroRNA-21 expression and chemoresistance in hyaluronan/CD44-activated head and neck squamous cell carcinoma cells.

MicroRNAs are often associated with the pathogenesis of many cancers, including head and neck squamous cell carcinoma (HNSCC). In particular, microRNA-21 (miR-21) appears to have a critical role in tumor cell survival, chemoresistance and HNSCC progression. In this study, we investigated matrix hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with the stem cell markers, Nanog and Stat-3, in HNSCC cells (HSC-3 cells). Our results indicate that HA binding to CD44 promotes Nanog-Stat-3 (also tyrosine phosphorylated Stat-3) complex formation, nuclear translocation and transcriptional activation. Further analyses reveal that miR-21 is controlled by an upstream promoter containing Stat-3 binding site(s), while chromatin immunoprecipitation assays demonstrate that stimulation of miR-21 expression by HA/CD44 signaling is Nanog/Stat-3-dependent in HNSCC cells. This process results in a decrease of a tumor suppressor protein (PDCD4), and an upregulation of i nhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in HSC-3 cells. Treatment of HSC-3 cells with Nanog- and/or Stat-3-specific small interfering RNAs effectively blocks HA-mediated Nanog-Stat-3 signaling events, abrogates miR-21 production and increases PDCD4 expression. Subsequently, this Nanog-Stat-3 signaling inhibition causes downregulation of survival protein (IAP) expression and enhancement of chemosensitivity. To further evaluate the role of miR-21 in tumor cell-specific functions, HSC-3 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and block its target functions. Our results demonstrate that anti-miR-21 inhibitor not only upregulates PDCD4 expression but also decreases IAP expression and enhances chemosensitivity in HA-treated HNSCC cells. Together, these findings indicate that the HA-induced CD44 interaction with Nanog and Stat-3 has a pivotal role in miR-21 production leading to PDCD4 reduction, IAP upregulation and chemoresistance in HNSCC cells. This novel Nanog/Stat-3 signaling pathway-specific mechanism involved in miR-21 production is significant for the formation of future intervention strategies in the treatment of HA/CD44-activated HNSCC.

Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185(HER2) and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth.

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. Delta N-Grb2 that has a deletion in the amino-terminal SH3 domain or Delta 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 Delta N-Grb2 (and to a lesser extent the Delta 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 co-activation of both Rac1 and Ras signaling that is required for HA-mediated human ovarian tumor progression.

CD44-mediated oncogenic signaling and cytoskeleton activation during mammary tumor progression.

CD44, a hyaluronan (HA) receptor, belongs to a family of transmembrane glycoproteins which exists as several isoforms. Cell surface expression of certain CD44 isoforms is closely correlated with the progression and prognosis of breast cancers. A number of angiogenic factors (e.g., VEGF and FGF-2) and matrix degrading enzymes (MMPs) are tightly complexed with CD44 isoforms, suggesting that they are involved in the onset of oncogenic signals required for breast tumor cell invasion and migration. Most importantly, interaction of extracellular matrix components (e.g., HA) with cells triggers the cytoplasmic domain of CD44 isoforms to bind its unique downstream effectors (e.g., the cytoskeletal protein ankyrin or various oncogenic signaling molecules-Tiam1, RhoA-activated ROK, c-Src kinase and p185HER2) and to coordinate intracellular signaling pathways (e.g., Rho/Ras signaling and receptor-linked/non-receptor-linked tyrosine kinase pathways), leading to a concomitant onset of multiple cellular functions (e.g., tumor cell growth, migration and invasion) and breast tumor progression.

A novel CD44 v3 isoform is involved in head and neck squamous cell carcinoma progression.

OBJECTIVES: CD44 comprises a family of isoforms involved in tumorigenesis. Here we investigate the role of CD44 isoforms in head and neck squamous cell carcinoma (HNSCC) progression. MATERIALS AND METHODS: HNSCC specimens underwent reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot analysis. After surface biotinylation, FaDu (hypopharyngeal HNSCC) and CD44v3-transfected COS-7 cells were CD44 antibody-precipitated and compared by Western blot analysis. FaDu cells underwent double immunofluorescence staining and growth assays. RESULTS: Southern blot analysis suggested differential CD44v3 isoform expression in tumor and normal tissue. Cloning and sequencing revealed 2 novel CD44v isoforms. Western blot analysis suggested CD44v3 expression in COS-7 transfectants and FaDu. Double immunofluorescence staining revealed co-localization of CD44v3 and actin in FaDu projections. Anti-CD44v3 antibody decreased FaDu growth. CONCLUSION: HNSCC tissue and FaDu appear to express CD44v3 isoforms. These isoforms may promote tumorigenesis. CLINICAL SIGNIFICANCE: CD44v3 isoforms may be effective tumor markers and targets for HNSCC therapy.

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 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 HA-dependent tumor cell migration, which may be required for human ovarian cancer progression.

Selective down-regulation of IP(3)receptor subtypes by caspases and calpain during TNF alpha -induced apoptosis of human T-lymphoma cells.

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP(3)R) [IP(3)-gated Ca(2+)channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP(3)R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca(2+)channel properties in an IP(3)-dependent manner. We have also demonstrated that TNFalpha promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca(2+)response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFalpha, several IP(3)R subtype-related changes occur (e.g. proteolysis of IP(3)R subtypes, inhibition of IP(3)binding and impairment of IP(3)-mediated Ca(2+)flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFalpha-mediated perturbation of IP(3)R1 and IP(3)R2 (but not IP(3)R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFalpha on IP(3)R3 function. These findings suggest that IP(3)R1 and IP(3)R2 serve as cellular substrates for caspases, and IP(3)R3 is a substrate for calpain. We propose that the selective down-regulation of IP(3)R subtype-mediated Ca(2+)function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFalpha in human T-cells.

Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration.

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11-amino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKRS(727)L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1). Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged recombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1-ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.

Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration.

The adhesion molecule, CD44, interacts with ankyrin within its cytoplasmic domain and binds to hyaluronic acid (HA) at its extracellular domain. In this study, we focused on the functional domain in ankyrin (in particular, the ankyrin repeat domain [ARD]) responsible for CD44 binding and its role in regulating HA-mediated ovarian tumor cell function. Using recombinant fragments of ankyrin (e.g., ARD and subdomain 1 [S1, aa1-aa217], subdomain 2 [S2, aa218-aa381], subdomain 3 [S3, aa382-aa612], and subdomain 4 [S4, aa613-aa834]) and in vitro binding assays, we determined that the S2 but not S1, S3, or S4 of ARD is the primary ankyrin binding region for CD44. Microinjection of antiglutathione S-transferase (GST)-tagged S2 or GST-tagged ARD fusion protein into CD44-positive ovarian tumor cells (e.g., SKOV3 cell line) promotes ankyrin association with CD44 in plaque-like structures and membrane projections. Additionally, we demonstrated that transfection of SKOV3 cells with S2cDNA or ARD cDNA results in an upregulation of HA-mediated tumor cell migration. Taken together, we believe that the S2 of the ARD plays a pivotal role in the direct binding to CD44 and promotes the cytoskeleton activation required for HA-mediated function such as ovarian tumor cell migration.

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 NH(2)-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 vitro binding 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) = approximately 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.

Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells.

Metastatic breast tumor Met-1 cells express CD44v(3,8-10), 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 CD44v(3,8-10) and RhoA GTPases are physically associated in vivo, and that CD44v(3,8-10)-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 CD44v(3,8-10)-bound RhoA GTPase. Specifically, RhoA (complexed with CD44v(3, 8-10)) stimulates ROK-mediated phosphorylation of certain cellular proteins including the cytoplasmic domain of CD44v(3,8-10). Most importantly, phosphorylation of CD44v(3,8-10) 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 CD44v(3,8-10)-ankyrin interaction and RhoA-mediated oncogenic signaling required for membrane-cytoskeleton function and metastatic tumor cell migration.

A new CD44V3-containing isoform is involved in tumor cell growth and migration during human breast carcinoma progression.

CD44 isoforms belong to a family of cell adhesion molecules expressed on the cell surface of many tumor cells during human breast cancer progression. In this study we have analyzed the expression of CD44v3-containing isoforms [containing heparan sulfate addition sites for growth factor binding] in primary breast tumors, axillary nodal metastases and normal breast tissue. Using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot, cloning, nucleotide sequencing and RT-in situ-PCR analyses, we have found that at least two CD44v3-containing isoforms, including one new species of CD44v2,deltav3-10 (deltav3 defined as a v3 exon lacking the first 24 base pairs) and another previously reported CD44v3,8-10 are preferentially expressed in human primary breast tumor and axillary nodal metastases but not in normal breast tissues. These finding suggest that these CD44v3-containing isoforms are closely associated with breast cancer metastasis.

CD44 isoform-cytoskeleton interaction in oncogenic signaling and tumor progression.

CD44, a major hyaluronan receptor, exists as several isoforms and is widely distributed in different cells and tissues. The isoforms of CD44, such as CD44s (the standard form), CD44E (the epithelial form) and CD44v (variant isoforms) (arise from differential splicing of one to ten (or eleven) variable exons that encode portions of the membrane proximal extracellular domain. The molecular diversity of CD44 isoforms is further compounded by differential biosynthetic processes and post-translational modifications [e.g. N-/O-glycosylation or glycosaminoglycan (GAG) addition]. This structural arrangement, which occurs within either the invariant region or the extracellular domain of the variant region, is important for CD44-mediated communication between extracellular matrix materials [ECM-hyaluronic acid (HA), collagen and fibronectin] and intracellular protein components (e.g cytoskeletal proteins and various regulatory enzymes). The 15 amino acid sequence [e.g. NSGNGAVEDRKPSGL (in human) or NGGNGTVEDRKPSEL (in mouse)] residing in the cytoplasmic domain of CD44 isoforms is the ankyrin-binding domain of this family of transmembrane glycoproteins. Biochemical analyses plus in vitro mutagenesis indicate that the ankyrin-binding domain is required for CD44-mediated "outside-in" and "inside-out" cell activation events. Furthermore, CD44s-cytoskeleton interaction is tightly coupled with signal transducing molecules (e.g. p185HER2 or Src kinases) during oncogenic signaling. Moreover, the transmembrane linkage between CD44v isoforms (CD44v10 and CD44v3) and the cytoskeleton up-regulates invasive and metastatic-specific tumor phenotypes [e.g. matrix degradation (MMPs) activities, tumor cell invasion and migration]. These findings strongly suggest that the interaction between CD44 isoforms and the cytoskeleton plays a pivotal role in the onset of oncogenesis and tumor progression.

CD44v(3,8-10) 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 approximately 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.

The ankyrin-binding domain of CD44s is involved in regulating hyaluronic acid-mediated functions and prostate tumor cell transformation.

CD44 isoforms, such as CD44s (the standard form), contain at least one ankyrin-binding site within the 70-amino acid (aa) cytoplasmic domain and several hyaluronic acid (HA)-binding sites within the extracellular domain. To study the role of CD44s-ankyrin interaction in regulating human prostate tumor cells, we have constructed several CD44s cytoplasmic deletion mutants that lack the ankyrin-binding site(s). These truncated cDNAs were stably transfected into CD44-negative human prostate tumor cells (LNCaP). Our results indicate that a critical region of 15-amino acids (aa) between aa 304 and aa 318 of CD44s is required for ankyrin binding. Biochemical analyses, using competition binding assays with a synthetic peptide containing the 15 aa between aa 304 and aa 318 (NSGNGAVEDRKPSGL), further support the conclusion that this region contains the ankyrin-binding domain of CD44s. Deletion of this 15-aa ankyrin-binding sequence from CD44s results in a drastic reduction of HA-mediated binding/cell adhesion, Src p60 kinase(s) interaction and anchorage-independent growth in soft agar. These findings suggest that the binding of cytoskeletal proteins, such as ankyrin, to the cytoplasmic domain of CD44s plays a pivotal role in regulating HA-mediated functions as well as Src kinase activity and prostate tumor cell transformation.

Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell activation.

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.

Coexpression of CD44 variant (v10/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis.

CD44 is the major hyaluronan cell surface receptor and functions as an adhesion molecule in many different cell types, including human breast epithelial cells. The coexpression of certain CD44 variants (CD44v), such as CD44v (v10/ex14), with CD44s (standard form) appears to be closely associated with human breast tumor metastasis. In this study we have established a stable transfection of CD44v (v10/ex14) cDNA into nontumorigenic human breast epithelial cells (HBL100) which contain endogenous CD44s. Our results indicate that coexpression of both CD44v (v10/ex14) and CD44s alters the following important biological properties of these cells: 1) there is a significant reduction in hyaluronic acid (HA)-mediated cell adhesion; 2) there is an increased migration capability in collagen-matrix gel; and 3) these cells constitutively produce certain angiogenic factors and effectively promote tumorigenesis in athymic nude mice. These findings suggest that coexpression of CD44v (v10/ex14) and CD44s may trigger the onset of cell transformation required for breast cancer development.

Involvement of CD44 and the cytoskeletal linker protein ankyrin in human neutrophil bacterial phagocytosis.

The leukocyte CD44 and CD45 cell surface receptors are associated via the linker proteins ankyrin and fodrin with the cytoskeleton, which itself is important in immune cell functions such as adherence, chemotaxis, and phagocytosis. The effects of rat antihuman CD44 and CD45 monoclonal antibodies on phagocytosis of fluoresceinated heat-killed Staphylococcus aureus 502A by normal human neutrophils (PMNs) during 2 hr incubation in RPMI-1640 was studied via flow cytometry and confocal microscopy. Flow cytometry was performed using an excitation wavelength of 488 nm, fluorescence being measured at 515-560 nm on 50,000 PMNs per sample. Confocal microscopy was performed on samples after further incubation with rhodamine-conjugated antiankyrin. Anti-CD44 resulted in an increase of 27-31% compared to control (P = 0.004) in the proportion of PMNs fluorescing, an increase of 17-24% (P = 0.001) in mean intracellular fluorescence per PMN, and an increase in total PMN fluorescence of 50-58% compared to control (P < 0.001). In contrast, anti-CD45 had little effect on phagocytosis. Colchicine (a microtubule-disrupting agent) enhanced, whereas cytochalasin-D (a microfilament inhibitor) inhibited bacterial phagocytosis; cytochalasin-D completely abrogated the effect of anti-CD44 on this PMN function. Hyaluronic acid augmented phagocytosis by an increment similar to that observed with anti-CD44. Two-color flow cytometry and confocal microscopy demonstrated that ankyrin always colocalized with ingested fluorescein isothiocyanate (FITC)-labeled bacteria. These data strongly suggest that CD44 is involved in bacterial phagocytosis, provide further evidence of CD44 receptor linkage to cytoskeletal elements in human leukocytes, and suggest that ankyrin has a significant role in the transport of phagosomes.

The cell adhesion molecule, GP116, is a new CD44 variant (ex14/v10) involved in hyaluronic acid binding and endothelial cell proliferation.

In this study we have found that endothelial cells from different origins all contain a CD44-related transmembrane glycoprotein, named GP116. Using a bovine aortic endothelial cell line and a standard pulse-chase protocol, we show that GP116 is synthesized as a 52-kDa nascent polypeptide precursor (p52) which is processed to GP116 as follows, p52 --> p63/65 --> p82 --> p100 --> GP116. GP116 contains approximately 8 N- and approximately 11 O-linked oligosaccharide chains (but lacks glycosaminoglycans) and interacts directly with the cytoskeletal protein, ankyrin, both in vitro (Kd approximately 1.2 nM) and in vivo. The results of GP116 amino acid composition, reverse transcriptase-polymerase chain reaction, Southern blot, Northern blot, cloning, and sequence analyses indicate that endothelial cells express this new CD44 variant that contains an exon having significant homology with human CD44 exon 14 (ex14/v10). GP116, designated as CD44 (ex14/v10), has been shown to be a major hyaluronic acid (HA) receptor (Kd approximately 0.5-0.8 nM) responsible for cell adhesion. Most importantly, we have found that the interaction between CD44(ex14/v10) and HA or a small fragment of HA (10-15 disaccharide units) induces a mitogenic response in endothelial cells. These findings suggest that this CD44 variant plays an important role in regulating endothelial cell proliferation.

Fatty acid unsaturation increases expression and capping of murine lymphocyte CD44 and CD45.

We studied the effect of incubating murine lymphocytes with cis-unsaturated fatty acids on expression and capping of CD44 and CD45. Lymphocytes were incubated with stearic (18:0) or oleic (18:1 omega-9) acid bound to bovine serum albumin (BSA). After incubation with rat anti-CD44 or anti-CD45 monoclonal antibodies and then with fluorescent-labeled anti-rat antibody, mean fluorescence intensity (FI) was measured by using flow cytometry. Capping was measured after warning and fixation in paraformaldehyde. Steady-state fluorescence anisotropy (rs) was measured after the cells had been incubated with trimethylammoniumdiphenylhexatriene. Incubation with oleic acid, but not stearic acid or BSA alone, was associated with an increase in FI of CD44. Expression of CD45, however, was increased by both stearic and oleic acids to the same degree over BSA controls. CD44 and CD45 capping were both increased by incubation with oleic acid. Rs was decreased in cells incubated with oleic acid, suggesting an increase in membrane fluidity. We conclude that incubation with oleic acid increases expression of CD44 and increases capping of both CD44 and CD45. These findings were confirmed in feeding experiments, in which rs was reduced and CD44 capping increased by polyunsaturated fatty acid diets.