Margot Zöller

A Complex of EpCAM, Claudin-7, CD44 Variant Isoforms, and Tetraspanins Promotes Colorectal Cancer Progression

High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. Expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. Expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7–associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation. (Mol Cancer Res 2007;5(6):553–67)

CD44 supports T cell proliferation and apoptosis by apposition of protein kinases

T cell activation is supposed to require two signals via the TCR and a co‐stimulatory molecule. However, the signaling cascade of co‐stimulatory molecules has remained elusive. Here we provide evidence that CD44, which is constitutively associated with lck and fyn, supports proliferation as well as apoptosis mainly, if not exclusively, by enhancing signal transduction via the TCR/CD3 complex. Antigenic stimulation of a T helper line in the presence of a CD44 receptor globulin was accompanied by a significant decrease in IL‐2 production. To evaluate the underlying mechanism, CD44 was cross‐linked via an immobilized antibody (IM‐7). Cross‐linking of CD44 induces proliferation of peripheral T cells and apoptosis of thymocytes and a T helper line in the presence of subthreshold levels of anti‐CD3. Several proteins are rapidly tyrosine phosphorylated; erk and c‐jun are strongly activated; expression of CD69 and CD25 is up‐regulated on mature T cells; and expression of CD95 and CD95L is up‐regulated on the T helper line. All these phenomena become less dependent of CD44 in the presence of high amounts of anti‐CD3. Furthermore, cross‐linking of CD44 is only effective when supporting co‐localization of CD44 with the TCR/CD3 complex, since mixtures of beads coated with either anti‐CD3 (low dose) or anti‐CD44 do not induce T cell activation. These findings imply the rearrangement of adhesion molecules with apposition of protein kinases as a critical event for the initiation of signaling via the TCR/CD3 complex.

Requirement for CD44 in proliferation and homing of hematopoietic precursor cells.

The hematopoietic form of the adhesion molecule CD44 is known to be involved in lymphocyte maturation and homing. To define lineage and stage of maturation, which requires expression of CD44, murine long‐term bone marrow cultures were established. Stroma formation and proliferation of early as well as committed erythroid, myeloid, and lymphoid progenitors were evaluated under the influence of monoclonal anti‐CD44 antibodies. Although the formation of stromal elements was not affected, formation of cobblestone areas was completely abolished. [3H]thymidine suicide confirmed that anti‐CD44 treatment interfered with the proliferation of early and committed hematopoietic progenitor cells. In addition, homing and seeding of bone marrow cells was impaired by anti‐CD44. The data are indicative of a dual functional mode of CD44 in adhesion and proliferation of hematopoietic progenitors and confirm an essential requirement for the molecule in early stages of hematopoiesis. J. Leukoc. Biol. 60: 579–592; 1996.

EXPRESSION OF CD44 SPLICE VARIANTS IN SQUAMOUS EPITHELIA AND SQUAMOUS CELL CARCINOMAS OF THE HEAD AND NECK

Splice variants of the adhesion molecule CD44 have been described as essential for the lymphatic spread of rat tumour cells and are claimed to be involved in the metastatic spread of several human tumours. Immunohistochemistry has been used to analyse the expression pattern of CD44 standard (CD44s) and variant (CD44v) isoforms in normal and dysplastic squamous epithelia, as well as in primary and metastatic squamous cell carcinomas (SCCs), which spread predominantly by way of the lymphatic system. Frozen sections of squamous epithelia and of squamous cell carcinomas were stained with a panel of monoclonal antibodies recognizing epitopes of CD44s as well as of the variant exons v5, v6, v7, v7–v8, and v10. The stratum basale and stratum suprabasale of squamous epithelia stained with all antibodies; the stratum spinosum stained with anti‐CD44v5, anti‐CD44v6, anti‐CD44v7–8 and anti‐CD44v10; the lower layers of the stratum corneum stained with anti‐CD44v5. This expression profile was seen in epithelia of the lip, the tongue, the gingiva, the hard palate, the floor of the mouth, the buccal mucosa, and the pharynx. The same pattern of expression was also noted in dysplastic epithelia, but expression of the variant exons v7, v8, and v10 was significantly downregulated in primary squamous cell carcinomas and was not detected at all in the majority of metastasis‐derived specimens. Expression of CD44v5 and CD44v6, on the other hand, was mainly unaltered. Thus, epithelial cell layers representing different stages of differentiation express distinct sets of CD44 variant isoforms, where especially exons v8–v10 might be required for the maintenance of the structural integrity of squamous epithelium. Downregulation of these exons on tumour cells could indicate that they are irrelevant for tumour progression or may even hamper infiltration of surrounding tissue or of lymphatics.

The Importance of Myeloid-Derived Suppressor Cells in the Regulation of Autoimmune Effector Cells by a Chronic Contact Eczema

Induction of a chronic eczema is a most efficient therapy for alopecia areata (AA). We had noted a reduction in regulatory T cells during AA induction and wondered whether regulatory T cells may become recruited or expanded during repeated skin sensitization or whether additional regulatory cells account for hair regrowth. AA could not be cured by the transfer of CD4+CD25high lymph node cells from mice repeatedly treated with a contact sensitizer. This obviously is a consequence of a dominance of freshly activated cells as compared with regulatory CD4+CD25+ T cells. Instead, a population of Gr-1+CD11b+ cells was significantly increased in skin and spleen of AA mice repeatedly treated with a contact sensitizer. Gr-1+CD11b+ spleen cells mostly expressed CD31. Expression of several proinflammatory cytokines as well as of the IFN-γ receptor and the TNF receptor I were increased. Particularly in the skin, Gr-1+ cells expressed several chemokines and CCR8 at high levels. Gr-1+CD11b+ cells most potently suppressed AA effector cell proliferation in vitro and promoted partial hair regrowth in vivo. When cocultured with CD4+ or CD8+ cells from AA mice, the Gr-1+CD11b+ cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein expression in AA T cells, apoptosis induction remained unaltered. Instead, ζ-chain expression was strongly down-regulated, which was accompanied by a decrease in ZAP70 and ERK1/2 phosphorylation. Thus, a chronic eczema supports the expansion and activation of myeloid suppressor cells that, via ζ-chain down-regulation, contribute to autoreactive T cell silencing in vitro and in vivo.

In Vitro and In Vivo Induction of a Th Cell Response Toward Peptides of the Melanoma-Associated Glycoprotein 100 Protein Selected by the TEPITOPE Program

The melanoma-associated Ag glycoprotein 100 was analyzed by the T cell epitope prediction software TEPITOPE. Seven HLA-DR promiscuous peptides predicted with a stringent threshold were used to load dendritic cells (DC), and induction of a proliferative response was monitored. PBMC of all nine donors including two patients with malignant melanoma responded to at least one of the peptides. The proliferative response was defined as a Th response by the selective expansion of CD4+ cells, up-regulation of CD25 and CD40L, and IL-2 and IFN-γ expression. Peptide-loaded DC also initiated a T helper response in vivo (i.e., tumor growth in the SCID mouse was significantly retarded by the transfer of PBMC together with peptide-loaded DC). Because the use of the TEPITOPE program allows for a prediction of T cell epitopes; because the predicted peptides can be rapidly confirmed by inducing a Th response in the individual patient; and because application of peptide-loaded DC suffices for the in vivo activation of helper cells, vaccination with MHC class II-binding peptides of tumor-associated Ags becomes a feasible and likely powerful tool in the immunotherapy of cancer.

CD44v10 expression in the mouse and functional activity in delayed type hypersensitivity.

We have described recently that expression of CD44 exon v10 (CD44v10) is down‐regulated upon metastasis of squamous cell carcinoma, whereas it is up‐regulated in skin metastases of malignant melanoma. The striking regulation of CD44v10 prompted us to generate a murine CD44v10‐specific monoclonal antibody to define expression and possible functions of this particular CD44 variant isoform. In the mouse, expression of exon v10 was restricted to basal layers of the epidermis and squamous epithelium of the oral cavity, the esophagus, the omasum, glandular epithelium of the submandibular and the uterine gland, as well as subpopulations of bone marrow cells and activated lymphocytes. Expression started late during development, e.g., was not observed before day 16 of gestation and there was no evidence for developmental regulation of CD44v10 expression. Functional in vivo studies revealed that anti‐CD44v10 had no effect on wound healing but inhibited edema and granuloma formation in delayed type hypersensitivity (DTH). Furthermore, lymphocyte‐monocyte interactions could be inhibited by anti‐CD44v10. Because a CD44v10 transfected tumour line did not show any distinct pattern of cell‐matrix or cell‐cell adhesion, the data point toward an involvement of CD44v10 in cell migration, possibly by acting as a target structure for cytokines/chemokines provided by the contacted partner cell. J. Cell. Physiol. 171:305–317, 1997.

Prophylactic tumor vaccination: comparison of effector mechanisms initiated by protein versus DNA vaccination.

Clinical success in tumor vaccination frequently does not reach expectation. Since vaccination protocols are quite variable, we used the murine renal cell carcinoma line RENCA transfected with the lacZ gene (RENCA-β-gal) to compare the efficacy of two different vaccination strategies or their combination and to elaborate on the underlying mechanisms. BALB/c mice were vaccinated either with naked lacZ DNA or with attenuated Salmonella typhimurium transformed with lacZ DNA or with dendritic cells (DC) loaded with the β-galactosidase protein or mice were vaccinated with both DNA and protein. Although all regimens led to a prolongation of survival time, oral vaccination with transfected S. typhimurium followed by i.v. transfer of protein-loaded DC provided the optimal schedule. In this setting, >50% of mice remained tumor free after challenge with 10 times the lethal tumor dose of RENCA-β-gal. As explored in transfer experiments, the superior efficacy of combining DNA and protein vaccination is due to the facts that 1) optimal protection depends on both activated CD4+ and CD8+ cells and 2) CD8+ CTL are most strongly activated by vaccination with transformed Salmonella, whereas vaccination with protein-loaded DC is superior for the activation of Th. The latter induced sustained activation of CTL and recruitment of nonadaptive defense mechanisms. The data demonstrate the strength of DNA vaccination, particularly by the oral route, and provide evidence that a combined treatment with protein-loaded DC can significantly increase the therapeutic efficacy.

Ly6 family member C4.4A binds laminins 1 and 5, associates with galectin-3 and supports cell migration.

C4.4A is a member of the Ly6 family, with low homology to uPAR. It has been detected mainly on metastasizing carcinoma cells and proposed to be involved in wound healing. So far, C4.4A has been observed as an orphan receptor, and its functional activity has not been explored. Using recombinant rat C4.4A (rrC4.4A) made in a eukaryotic expression system, we demonstrate by immunohistology that C4.4A ligands are strongly expressed in tissues adjacent to squamous epithelia of, e.g., tongue and esophagus, the expression pattern partly overlapping with laminin (LN) and complementing the C4.4A expression that is found predominantly on the basal layers of squamous epithelium. ELISA screening of several components of the extracellular matrix revealed selective binding of rrC4.4A to LN1 and LN5 and that transfection of the BSp73AS tumor line with C4.4A cDNA (BSp73AS‐1B1) promoted LN1 and LN5 binding. Binding of BSp73AS‐1B1 to LN5 and, less markedly, LN1 induced spreading, lamellipodia formation and migration. C4.4A also associates with galectin‐3 in nontransformed tissues and tumor lines. There is evidence that the association of C4.4A with galectin‐3 influences LN adhesion. C4.4A was described originally as a metastasis‐associated molecule. Our findings that LN1 and LN5 are C4.4A ligands, that galectin‐3 associates with C4.4A and that C4.4A ligand binding confers a migratory phenotype are well in line with the supposed metastasis association.

Cloning of the human homologue of the metastasis-associated rat C4.4A

We have previously described a rat metastasis-associated molecule, C4.4A, which has some common features with the uPAR. Because of its restricted expression in non-transformed tissues a search for the human homologue became of interest. Human C4.4A was cloned from a placental cDNA library. As in the rat, the human uPAR and the human C4.4A genes appear to belong to the same family. Both genes are located on chromosome 19q13.1-q13.2 and both molecules have a glycolipid anchor site and are composed of three extracellular domains. Only domains one and two of the human C4.4A and the uPAR protein show a significant degree of identity. Expression of the human C4.4A was observed by RT-PCR and Northern blotting in placental tissue, skin, esophagus and peripheral blood leukocytes, but not in brain, lung, liver, kidney, stomach, colon and lymphoid organs. Yet, tumors derived from the latter tissues frequently contained C4.4A mRNA. As demonstrated for malignant melanoma, C4.4A mRNA expression correlated with tumor progression. While nevi were negative and only a minority of primary malignant melanoma expressed C4.4A, all metastases were C4.4A-positive. Taking into account the high degree of homology between rat and human C4.4A, the conformity of the expression profiles and the association of rat C4.4A with tumor progression, human C4.4A might well become a prognostic marker and possibly a target of therapy.