Here, we report unbiased screens for genes expressed in metastatic tumor cells that are associated with cell motility. These screens identified Ier2, an immediate early gene of unknown function, as potentially having a role in tumor cell motility and metastasis. Knockdown of Ier2 in 3T3 fibroblasts inhibited their motility upon relief of contact inhibition in monolayer wounding assays. Furthermore, ectopic Ier2 expression promoted the motility and invasiveness of poorly metastatic 1AS pancreatic tumor cells in vitro. Relief of contact inhibition was associated with translocation of the Ier2 protein from the cytoplasm to the nucleus in both 3T3 fibroblasts and 1AS tumor cells. Importantly, ectopic Ier2 expression in 1AS cells stimulated metastasis formation when cells were implanted into experimental animals. Furthermore, we found elevated Ier2 expression in a wide variety of human tumor types. This correlated with poor metastasis-free and overall survival in patients with colorectal adenocarcinomas. Together, these data reveal Ier2 as a new player in the regulation of tumor progression and metastasis, and suggest that Ier2 may be useful prognostically and therapeutically in the management of cancer.

Cell motility is an essential element of tumor dissemination, allowing organ infiltration by cancer cells. Using mouse LB lymphoma cells transfected with standard CD44 (CD44s) cDNA (LB-TRs cells) or with the alternatively spliced CD44 variant CD44v4-v10 (CD44v) cDNA (LB-TRv cells), we explored their CD44-dependent cell migration. LB-TRv cells, but not LB-TRs or parental LB cells, bound soluble hyaluronic acid (HA) and other glycosaminoglycans (GAGs), and exclusively formed, under physiological shear force, rolling attachments on HA substrate. Furthermore, LB-TRv cells, but not LB-TRs cells or their parental LB cells, displayed accelerated local tumor formation and enhanced accumulation in the peripheral lymph nodes after s.c. inoculation. The aggressive metastatic behavior of i.v.-injected LB-TRV cells, when compared with that of other LB-transfectants, is attributed to more efficient migration to the lymph nodes, rather than to local growth in the lymph node. Injection of anti-CD44 monoclonal antibody or of the enzyme hyaluronidase also prevented tumor growth in lymph nodes of BALB/c mice inoculated with LB-TRv cells. The enhanced in vitro rolling and enhanced in vivo local tumor growth and lymph node invasion disappeared in LB cells transfected with CD44v cDNA bearing a point mutation at the HA binding site, located at the distal end of the molecule constant region. These findings show that the interaction of cell surface CD44v with HA promotes cell migration both in vitro and in vivo, and they contribute to our understanding of the mechanism of cell trafficking, including tumor spread.

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.

The CD44v6 epitope has been widely reported to be expressed in human mammary carcinomas, yet its prognostic significance is controversial and its function in mammary tumors and mammary glands is unknown. To begin to resolve these issues, we analysed in detail the normal postnatal expression patterns and regulation of the CD44v6 epitope in murine mammary glands. We demonstrate that significant CD44v6 epitope expression is first seen during puberty, and that after puberty CD44v6 epitope expression follows the estrous cycle. CD44v6 epitope expression is observed in the myoepithelium and also less widely in luminal epithelial cells. During lactation, CD44v6 epitope expression is turned off and reappears during involution. The CD44 variant isoform bearing the v6 epitope is CD44v1-v10. Using HC11, a mammary epithelial cell line with stem cell characteristics, and facilitated by the cloning of the murine CD44 promoter, we show that growth factors and hormones which regulate ductal growth and differentiation modulate CD44 transcription. Together our data suggest that the CD44v6 epitope is expressed in mammary epithelial stems cells and in lineages derived from these cells, and that CD44v6 expression is regulated in part by hormones and growth factors such as IGF-1 and EGF which regulate the growth and differentiation of the mammary epithelium. The function of these same growth factors and hormones is often perturbed in mammary carcinomas, and we suggest that CD44v6 expression in tumors reflects this perturbation. We conclude that the expression of the CD44v6 epitope observed in some mammary tumors reflects the stem cell origin of breast tumors, and that whether or not the CD44v6 epitope is expressed in a mammary tumor is determined by the differentiation status of the tumor cells.

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.

During inflammation, activated monocytes (Mo) migrate into tissues where they interact with extracellular matrix components such as hyaluronate (HA), produced in high amounts at inflammatory sites. We determined whether Mo that had invaded sites of cutaneous inflammation bind HA and express the putative HA receptors CD44 isoforms, ICAM-1, or receptor for hyaluronate-mediated motility (RHAMM). In cutaneous inflammation, activated infiltrating Mo displayed high HA avidity and expressed epitopes encoded by CD44s, CD44 variant exons v3, v4, v5, v6, v7, and v9, and ICAM-1, but not RHAMM. We further investigated how activation affects the avidity of Mo for HA and which receptors were responsible for such binding. Mo freshly purified from human peripheral blood bound little HA and expressed CD44s but no epitopes encoded by CD44v exons, ICAM-1, or RHAMM. During short-term tissue culture, Mo upregulated their HA avidity and expression of ICAM-1, CD44s, and epitopes encoded by CD44v, all of which were further augmented by IFN-gamma or lipopolysaccharide, whereas RHAMM was not detectable. Thus in vitro activated Mo resembled Mo that had migrated to inflammatory sites in vivo. Lipolysaccharide or IFN-gamma-induced HA binding was inhibited by more than 90% with monoclonal antibodies directed against N-terminal HA binding domains of CD44s, but not by monoclonal antibodies against CD44v epitopes or ICAM-1. In conclusion, we show that upon in vitro or in vivo activation, Mo enhance their capacity to bind HA. This is critically dependent upon the expression ofCD44s epitopes. Regulated CD44-HA interactions may be important for the ability of Mo to migrate into and within sites of inflammation and for Mo effector functions.

CD44 is important during myelopoiesis, although the contributions of variant CD44 proteins are unclear. We show here that in human long-term bone marrow culture antibodies recognizing a CD44 NH2-terminal epitope (mab 25-32) or a CD44v6 epitope (mab VFF18) inhibit myelopoiesis. However, mab 25-32 but not mab VFF18 affects myeloid colony formation. These data suggest that an early precursor cell compartment is the target for the 25-32 antibody, whereas the mab VFF18 targets later stages in myelopoiesis. Since the bulk of hemopoietic precursor cells are negative for the v6 epitope and only a minor subset of myeloid cells express the v6 epitope, we have used several human myeloid progenitor cell lines to unravel the function of different CD44 proteins. These cell lines produce variant CD44 proteins, predominantly a new variant CD44v4-v10, when stimulated towards myeloid differentiation. Features that can be acquired by the expression of CD44v4-v10 are an increased hyaluronate (HA) and a de novo chondroitin sulphate A (CS-A) binding. Although, the expression of CD44v4-v10 per se is necessary for HA and CS-A binding, the protein backbone seems to require appropriate glycosylation. HA binding results in CD44-mediated cellular self-aggregation and adhesion to the stromal cell line MS-5. In summary, our data suggest that different CD44 proteins are important for at least two different steps in myelopoiesis.

Constitutive expression of a rat CD44 variant isoform, rCD44v4-v7, on murine T cells accelerates immune responsiveness. Because prolonged immunodeficiency can be a major drawback in allogeneic bone marrow transplantation, we considered it of special interest to see whether repopulation of lethally irradiated syngeneic and allogeneic mice may be influenced by constitutive expression of the rCD44v4-v7 transgene. When lethally irradiated syngeneic and allogeneic mice were reconstituted with bone marrow cells (BMC) from rCD44v4-v7 transgenic (TG) or nontransgenic (NTG) mice, the former had a clear repopulation advantage: thymocytes expanded earlier after reconstitution and, as a consequence, higher numbers of lymphocytes were recovered from spleen and lymph nodes. Lymphocytes also displayed functional activity in advance to those from mice reconstituted with BMC from NTG mice. Most importantly, after the transfer of BMC from TG mice into an allogeneic host, the frequency of host-reactive T cells decreased rapidly. Apparently, this was due to accelerated induction of tolerance. Because these effects were counterregulated by an rCD44v6-specific antibody, it is likely that they could be attributed to the rCD44v4-v7 TG product. Thus, expression of a CD44 variant isoform at high levels facilitated reconstitution with allogeneic BMC by accelerated establishment of tolerance and the regaining of immunocompetence.

Isoforms of the glycoprotein CD44 are cell surface receptors for the glycosaminoglycan hyaluronate. They have been implicated in many biological processes, but their function in these is poorly understood and cannot be explained solely by hyaluronate binding. In the present work we examine the ligand binding properties of alternatively spliced CD44 variant isoforms which are functionally involved in the immune system, embryonic development, and tumor behavior. We show that these isoforms bind directly to the purified glycosaminoglycans chondroitin sulfate, heparin, and heparin sulfate, in addition to being able to bind to hyaluronate. Binding to this extended repertoire of glycosaminoglycans by CD44 depends on the inclusion of peptide sequences encoded by the alternatively spliced exons v6 and v7, and occurs both when the CD44 is solubilized from the plasma membrane and when it is expressed on intact cells. A single point mutation in the most N-terminal hyaluronate binding motif of CD44 ablates both hyaluronate and chondroitin sulfate binding, suggesting that glycosaminoglycans are bound through a common motif, and that only one of the hyaluronate binding motifs is responsible for the majority of glycosaminoglycan binding by CD44 on the cell surface. Taken together, these observations indicate that alternative splicing regulates the ligand binding specificity of CD44 and suggest that structural changes in the CD44 protein have a profound effect on the range of ligands to which this molecule can bind with potentially wide-ranging functional consequences.

Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell‐type specific trans‐acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant‐exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans‐acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3′ to 5′ direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis‐acting recognition sites.

During the past decade, considerable advances have been made in our under­standing of the cellular and molecular biology of carcinogenesis. The analysis of the metastatic process, however, which is the chief contributor to cancer patient morbidity, has met with various obstacles due to its complexity. As a result, we still know very little about how cancer cells spread. It is generally assumed that cancer cells randomly diversify during tumor progression and that clones of such cells with metastatic properties are selected. The process by which these cells then metastasize is believed to occur via several steps, each of which can be linked to the aquisition of new cellular functions (see, e.g., reviews by Hart et al. 1989; Kerbel 1990). For example, a stationary epithelial cell must first be con­verted into a mobile, nonadherent cancer cell which can invade subepithelial tissue. The cell must then move towards lymphatic and blood vessels, enter these vessels, and survive during their transport within the fluid medium. It must also colonize draining lymphatic tissue and further disseminate through efferent lymphatics into the blood stream, extravasate from blood vessels and, finally, preferentially settle in various distant locations.

Transgenic mice were generated which express CD44v4-v7 on the surface of 20-80% of their T cells, depending on which lymphoid organ the T cells were derived from. Soluble CD44(v4-v7) is present in their serum, showing that, like CD44s, CD44v can be shedded from the cell surface. The incidence of chemically induced lymphomas in these animals was not increased as compared to control mice, nor were the tumours more aggressive. The importance of CD44v in malignancies and during early stages of hemopoietic differentiation are discussed.

Splice variants of the cell surface glycoprotein CD44 (CD44v) have been implicated in the progression of various human tumors. In the present study, we have examined the expression pattern of a CD44v epitope encoded by the adjacent variant exons v7 and v8 during human cervical carcinogenesis, While only I/II normal cervical squamous epithelia was positive for this epitope by immunohistochemistry, 4/21 samples of low-grade squamous intra-epithelial lesions (LSIL), 17/35 samples of high-grade squamous intra-epithelial lesions (HSIL), 11/12 samples of the HSIL subgroup of carcinoma in site and 17/17 cases of invasive cervical carcinoma showed CD44v7/8 epitope expression. In addition to CD44 variant expression, we have analyzed 67 lesions for the presence of HPV16/18-DNA using PCR. Most of the samples expressing the v7/8 epitope were also HPV16-positive (29/32), whereas only 17/35 of the v7/8-negative samples were HPV16-positive. HPV18 DNA was found in only one invasive carcinoma, Our data suggest that high-risk HPV infection may precede CD44v7/8 expression and that the number of samples expressing the CD44 v7/8 epitope increases during carcinogenesis and reaches nearly 100% at the carcinoma in situ stage. This CD44 epitope could, therefore, serve as a diagnostic marker of cervical squamous cell carcinomas and as a possible target for CD44v7/8 epitope-directed therapies. (C) 1996 Wiley-Liss, Inc.

Splice variants of the glycoprotein CD44 (CD44v) that confer metastatic behavior to noninvasively growing rat tumor cells are transiently expressed on lymphocytes during activation. A mAb directed against an epitope encoded by CD44 exon v6 blocks both metastasis formations and lymphocyte activation, implicating CD44v in normal immune function. To explore the nature of this function of CD44v, transgenic mice were generated that constitutively express rat CD44v4-v7 on thymocytes and peripheral T cells. The number of lymphocytes as well as the distribution of lymphocyte subpopulations were similar in nontransgenic and rat CD44v4-v7 transgenic mice. T cells of the transgenic mice, however, responded faster to activating stimuli, particularly during primary stimulations with T cell mitogens and T-dependent Ags in vivo and in vitro. This accelerated response depended on the expression of the transgene product, since a rat CD44v6-specific Ab reverted the response profiles of the transgenic mice to those of nontransgenic mice. Since the transgene gained in vivo and in vitro functional activity only upon antigenic stimulation, it is likely that CD44 variant isoforms are involved in the process of signal transduction during lymphocyte activation.

Cell contact with the extracellular matrix component hyaluronic acid (HA) plays an important role in many developmental, physiological, and pathological processes, although the regulation of this contact is poorly understood. CD44 proteins carry an amino acid motif that mediates affinity to HA. Artificial clustering of the smallest 85-kD isoform of CD44 (CD44s) has previously been shown to promote binding of the protein to soluble HA (Lesley, J., R. Hyman, and P.W. Kincade. 1993. Adv. Immunol. 54:271-335; Persche, A., J. Lesley, N. English, I. Trowbridge, and R. Hyman. 1995. Eur. J. Immunol. 25:495-501). Here we show that in rat pancreatic carcinoma cells, splice variants of CD44 (CD44v), but not CD44s, form molecular aggregates in the plasma membrane. We demonstrate that reduction-sensitive dimerization of CD44v occurs, and also that larger aggregations of the protein can be stabilized by chemical cross-linking. Different CD44v proteins present on the same cell exclusively form homoaggregates. Molecular clustering does not require an intact cytoplasmic domain of the protein. The ability of cells to bind to soluble HA is upregulated more than one magnitude by the ectopic expression of CD44v4-v7, but only when the CD44v4-v7 protein forms intermolecular aggregates. Tunicamycin treatment inhibits HA binding by CD44v and at the same time destroys oligomerization. We propose that the regulation of clustering of CD44, mediated by factors including the presence of variant exons and glycosylation, allows cells in turn to regulate their HA binding properties.