Naoko Iida

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.

Hyaluronic Acid Synthase-1 Expression Regulates Bladder Cancer Growth, Invasion, and Angiogenesis through CD44

Hyaluronic acid (HA) promotes tumor metastasis and is an accurate diagnostic marker for bladder cancer. HA is synthesized by HA synthases HAS1, HAS2, or HAS3. We have previously shown that HAS1 expression in tumor tissues is a predictor of bladder cancer recurrence and treatment failure. In this study, we stably transfected HT1376 bladder cancer cells with HAS1-sense (HAS1-S), HAS1-antisense (HAS1-AS), or vector cDNA constructs. Whereas HAS1-S transfectants produced approximately 1.7-fold more HA than vector transfectants, HA production was reduced by approximately 70% in HAS1-AS transfectants. HAS1-AS transfectants grew 5-fold slower and were approximately 60% less invasive than vector and HAS1-S transfectants. HAS1-AS transfectants were blocked in G(2)-M phase of the cell cycle due to down-regulation of cyclin B1, cdc25c, and cyclin-dependent kinase 1 levels. These transfectants were also 5- to 10-fold more apoptotic due to the activation of the Fas-Fas ligand-mediated extrinsic pathway. HAS1-AS transfectants showed a approximately 4-fold decrease in ErbB2 phosphorylation and down-regulation of CD44 variant isoforms (CD44-v3, CD44-v6, and CD44-E) both at the protein and mRNA levels. However, no decrease in RHAMM levels was observed. The decrease in CD44-v mRNA levels was not due to increased mRNA degradation. Whereas CD44 small interfering RNA (siRNA) transfection decreased cell growth and induced apoptosis in HT1376 cells, HA addition modestly increased CD44 expression and cell growth in HAS1-AS transfectants, which could be blocked by CD44 siRNA. In xenograft studies, HAS1-AS tumors grew 3- to 5-fold slower and had approximately 4-fold lower microvessel density. These results show that HAS1 regulates bladder cancer growth and progression by modulating HA synthesis and HA receptor levels.

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. J. Cell. Physiol. 171:152–160, 1997.

Involvement of CD44 and its variant isoforms in membrane-cytoskeleton interaction, cell adhesion and tumor metastasis

SummaryCD44s (standard form of CD44) is a transmembrane glycoprotein whose external domain displays extracellular matrix adhesion properties by binding both hyaluronic acid (HA) and collagen. The cytoplasmic domain of CD44s interacts with the cytoskeleton by binding directly to ankyrin. It has been shown that post-translational modifications, such as phosphorylation (by protein kinase C), acylation (by acyl-transferase) and GTP-binding enhance CD44s interaction with cytoskeletal proteins. Most importantly, the interaction between CD44s and the cytoskeletal protein, ankyrin, is required for the modulation of CD44s cell surface expression and its adhesion function.Recently, a number of tumor cells and tissues have been shown to express CD44 variant (CD44v) isoforms. Using RT-PCR and DNA sequence analyses, we have found that unique CD44 splice variant isoforms are expressed in both prostate and breast cancer cell lines and carcinomas. Most importantly, intracellular ankyrin is preferentially accumulated underneath the patched/capped structures of CD44 variant isoform in both breast and prostate cancer cells attached to HA-coated plates. We propose that selective expression of CD44v isoforms unique for certain metastatic carcinomas and their interaction with the cytoskeleton may play a pivotal role in regulating tumor cell behavior during tumor development and metastasis.

The involvement of the cytoskeleton in regulating IP3 receptor-mediated internal Ca2+ release in human blood platelets.

In this study we have used saponin to permeabilize platelet membranes in order to test directly the involvement of IP3 in regulating internal Ca2+ release, and to measure IP3 binding to its receptor. Our results indicate that platelet vesicles release Ca2+ as early as 3 seconds after IP3 addition. Using [3H]IP3, we have found that platelets contain a single class of high affinity IP3 binding sites with a Kd of ∼0.20 (± 0.01) nM. Immuno‐blotting shows that platelets contain a 260 kDa polypeptide which shares immunological cross reactivity with brain IP3 receptor. Immunofluorescence staining data indicate that the IP3 receptor is preferentially located at the periphery of the platelet plasma membrane. Most importantly, both IP3 binding and IP3‐induced Ca2+ release activities are significantly inhibited by cytochalasin D (a microfilament inhibitor) and colchicine (a microtubule inhibitor). These findings suggest that the cytoskeleton is involved in the regulation of IP3 binding and IP3 receptor‐mediated Ca2+ release during platelet activation.

A new splice variant of the inositol-1,4,5-triphosphate (IP3) receptor

In this study we have identified a new splice variant of the IP3 receptor (IP3R) transcript in a number of mouse cell lines (e.g. mouse T-lymphoma cells, mouse splenic lymphocytes and mouse NIH 3T3 fibroblast cell lines) using the reverse transcriptase-polymerase chain reaction. This variant IP3 receptor (designated as IP3RV-S2, approximately 453 bp) is larger than the non-neuronal form (402 bp) but smaller than the neuronal form (522 bp) of the IP3 receptors. Nucleotide sequencing data indicate that this new isoform (IP3RV-S2) contains a 51 nucleotide insertion within the non-neuronal form of IP3R at the S2 splice site. During mitogenic stimulation by Con A, the ratio between IP3R (non-neuronal form) and IP3RV-S2 (variant isoform) in mouse splenic T-lymphocytes increases approximately 1.5-fold. The change in relative amounts of these two IP3 receptor isoforms during mitogenic-stimulation suggests that T-lymphocytes may have different requirements for the IP3 isoforms in order to control intracellular calcium mobilization. The selective expression of these two IP3R isoforms (IP3RV-S2 and non-neuronal IP3R) may be critically important for the onset of signal transduction and cell activation.