Rachid Marhaba

CD44 and EpCAM: Cancer-Initiating Cell Markers

Embryonic stem cells are immortal, can self renew, and differentiate into all cells of the body. The adult organism maintains adult stem cells in regenerative organs that can differentiate into all cells of the respective organ. Virchows hypothesis that cancer may arise from embryonic-like cells has received strong support, as it was demonstrated that tumors contain few cells, known as cancer stem or cancer-initiating cells (CIC), that account for primary and metastatic tumor growth. CIC are mostly defined by expression of CIC-markers that are associated and correlated with the potential of CIC to grow in xenogeneic mice. CIC marker profiles have been elaborated for many tumors, with several markers as CD24, CD44, CD133, CD166, EpCAM, and some integrins, being expressed by tumors of different histological type. Their function in promoting CIC maintenance and activity is largely unknown. The fate of stem cells, determined by their position, is minutely regulated by few adjacent cells creating a niche. CIC also require a niche, mostly for settlement and growth in distant organs. This so called pre-metastatic niche is initiated by the primary tumor before metastasizing cell arrival. How do CIC prepare the pre-metastatic niche? Cancer cells secrete a matrix that serves a cross-talk with surrounding tissues. Additionally, cancer cells can abundantly deliver exosomes, which function as long-distance intercellular communicators. Studies on a rat pancreatic adenocarcinoma support our hypothesis that tumor-derived matrix and exosomes are the main actors in forming the pre-metastatic niche with CIC markers being engaged in matrix preparation and/or exosome delivery.

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.

CD44 variant isoforms promote metastasis formation by a tumor cell-matrix cross-talk that supports adhesion and apoptosis resistance.

CD44 designates a large family of proteins with a considerable structural and functional diversity, which are generated from one gene by alternative splicing. As such, the overexpression of CD44 variant isoform (CD44v) has been causally related to the metastatic spread of cancer cells. To study the underlying mechanism, stable knockdown clones with deletion of exon v7 containing CD44 isoforms (CD44vkd) of the highly metastatic rat adenocarcinoma line BSp73ASML (ASMLwt) were established. ASML-CD44vkd clones hardly form lung metastases after intrafootpad application and the metastatic load in lymph nodes is significantly reduced. Rescuing, albeit at a reduced level, CD44v expression in ASML-CD44vkd cells (ASML-CD44vrsc) restores the metastatic potential. The following major differences in ASMLwt, ASML-CD44vkd, and ASML-CD44vrsc clones were observed: (a) ASMLwt cells produce and assemble a matrix in a CD44v-dependent manner, which supports integrin-mediated adhesion and favors survival. This feature is lost in the ASML-CD44vkd cells. (b) CD44v cross-linking initiates phosphatidylinositol 3-kinase/Akt activation in ASMLwt cells. Accordingly, apoptosis resistance is strikingly reduced in ASML-CD44vkd cells. The capacity to generate an adhesive matrix but not apoptosis resistance is restored in ASML-CD44vrsc cells. These data argue for a 2-fold effect of CD44v on metastasis formation: CD44v-mediated matrix formation is crucial for the settlement and growth at a secondary site, whereas apoptosis resistance supports the efficacy of metastasis formation. (Mol Cancer Res 2009;7(2):168–79)

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.

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.

In vivo CD44-CD49d complex formation in autoimmune disease has consequences on T cell activation and apoptosis resistance

CD44 is involved in leukocyte migration and activation and has recently been reported to contribute to leukocyte extravasation by associating with CD49d. We explored whether similar changes in CD44 activity are seen in vivo using murine alopecia areata (AA) as a chronic, organ‐related autoimmune disease model system. Expression of the activated, hyaluronan‐binding form of CD44, and of CD49d, was elevated in draining lymph node cells (LNC) of AA‐affected mice as compared to control mice. LNC of AA mice displayed increased motility, proliferative activity and apoptosis resistance, which were equally well inhibited by anti‐CD44 and anti‐CD49d. The latter is the sequelae of the association between CD44 and CD49d that is seen in activated lymphocytes. Significantly, due to CD44‐CD49d complex formation, CD44 gains access to focal adhesion kinase and CD49d gains access to CD44‐associated lck and ezrin, such that downstream kinases become activated via CD44 or CD49d engagement. Thus, by their association, CD44 and CD49d mutually avail themselves of the partners signaling pathways and the ligand binding of each one triggers signaling pathways of both. This strongly influences the lymphocytes’ activation state and function.

CD44v7 interferes with activation-induced cell death by up-regulation of anti-apoptotic gene expression

Blockade of CD44v7 was described to cure trinitrobenzene sulfonic acid‐induced colitis, a disease not developed by mice with targeted deletion of the CD44v7 exon. There was evidence for a reduction in activation‐induced cell death on lamina propria lymphocytes of control as compared with CD44v7‐deficient mice. To elucidate the mechanism underlying the relative apoptosis resistance of CD44v7‐competent as compared with CD44v7‐deficient lymphocytes, T cell activation and induction of apoptosis were analyzed on mesenteric lymph node cells and Peyer’s patch lymphocytes of CD44v7‐deficient and CD44v4‐v7‐transgenic mice, which overexpress rat CD44v4‐v7 on T lymphocytes. CD44v7 deficiency was characterized by an increase in the percentage of apoptotic cells after stimulation, increased numbers of CD95L‐ and CD152‐positive cells, low levels of the anti‐apoptotic proteins Bcl‐2 and Bcl‐Xl, and decreased phosphorylation of the pro‐apoptotic protein BAD. Also, lymphocytes from CD44v4‐v7‐transgenic mice displayed reduced levels of CD95L, low numbers of apoptotic cells, and constitutively elevated levels of Bcl‐Xl. When stimulating lymphocytes by CD3 cross‐linking, CD44v7 was not recruited toward the immunological synapse and preferentially associated with the cytoskeletal‐linker protein ezrin. Thus, as opposed to the CD44 standard isoform, CD44v7 does not function as an accessory molecule; instead, it supports survival of activated T cells by interfering with activation‐induced cell death.

Anti‐CD44‐mediated blockade of leukocyte migration in skin‐associated immune diseases

CD44 plays an important role in leukocyte extravasation, which is fortified in autoimmune diseases and delayed‐type hypersensitivity (DTH) reactions. There is additional evidence that distinct CD44 isoforms interfere with the extravasation of selective leukocyte subsets. We wanted to explore this question in alopecia areata (AA), a hair‐follicle centric autoimmune disease, and in a chronic eczema. The question became of interest because AA is treated efficiently by topical application of a contact sensitizer, such that a mild DTH reaction is maintained persistently. Aiming to support the therapeutic efficacy of a chronic eczema in AA by anti‐CD44 treatment, it became essential to control whether a blockade of migration, preferentially of AA effector cells, could be achieved by CD44 isoform‐specific antibodies. Anti‐panCD44 and anti‐CD44 variant 10 isoform (CD44v10) inhibited in vitro migration of leukocytes from untreated and allergen‐treated, control and AA mice. In vivo, both antibodies interfered with T cell and monocyte extravasation into the skin; only anti‐panCD44 prevented T cell homing into lymph nodes. Contributing factors are disease‐dependent alterations in chemokine/chemokine receptor expression and a blockade of CD44 on endothelial cells and leukocytes. It is important that CD44 can associate with several integrins and ICAM‐1. Associations depend on CD44 activation and vary with CD44 isoforms and leukocyte subpopulations. CD44 standard isoform preferentially associates with CD49d in T cells and CD44v10 with CD11b in monocytes. Accordingly, anti‐panCD44 and anti‐CD49d inhibit T cell, anti‐CD11b, and anti‐CD44v10 macrophage migration most efficiently. Thus, allergen treatment of AA likely can be supported by targeting AA T cells selectively via a panCD44‐CD49d‐bispecific antibody.

CD44 promotes progenitor homing into the thymus and T cell maturation

Regain of immunocompetence after myeloablation and bone marrow cell (BMC) reconstitution essentially depends on T progenitor homing into the thymus and intrathymic T cell maturation. CD44 facilitates progenitor homing and settlement in the bone marrow and is known as a T progenitor marker. In search for improving regain of immunocompetence after BMC reconstitution, we explored whether the CD44 standard (CD44 s) and/or variant isoforms CD44v6 and CD44v7 contribute to thymus repopulation and thymocyte maturation. Antibody‐blocking studies and cells/mice with a targeted deletion of CD44v6/7 or CD44v7 revealed that CD44s, but not CD44v6 and CD44v7, has a major impact on progenitor cell homing into the thymus. Instead, CD44v6 strengthens apoptosis resistance and expansion of early thymocytes. CD44v6‐induced apoptosis resistance, most strong in double‐negative (DN) thymocytes, is accompanied by Akt activation. CD44v6‐induced proliferation of DN cells proceeds via activation of the MAPK pathway. At later stages of T cell maturation, CD44 acts as an accessory molecule, initiating and supporting TCR/CD3 complex‐mediated signal transduction in double‐positive and single‐positive thymocytes. Thus, CD44 plays a major role in thymus homing. In addition, CD44v6 is important for survival and expansion of early thymocytes. These findings suggest that strengthening CD44v6 expression on lymphoid progenitors could well contribute to accelerated regain of immunocompetence.

Anti-CD44 induces apoptosis in T lymphoma via mitochondrial depolarization.

A blockade of CD44 can interfere with haematopoietic and leukemic stem cell homing, the latter being considered as a therapeutic option in haematological malignancies. We here aimed to explore the molecular mechanism underlying the therapeutic efficacy of anti‐CD44. We noted that in irradiated mice reconstituted with a bone marrow cell transplant, anti‐CD44 exerts a stronger effect on haematopoietic reconstitution than on T lymphoma (EL4) growth. Nonetheless, in the non‐reconstituted mouse anti‐CD44 suffices for a prolonged survival of EL4‐bearing mice, where anti‐CD44‐prohibited homing actively drives EL4 cells into apoptosis. In vitro, a CD44 occupancy results in a 2–4‐fold increase in apoptotic EL4 cells. Death receptor expression (CD95, TRAIL, TNFRI) remains unaltered and CD95 cross‐linking‐mediated apoptosis is not affected. Instead, CD44 ligation promotes mitochondrial depolarization that is accompanied by caspase‐9 cleavage and is inhibited in the presence of a caspase‐9 inhibitor. Apoptosis becomes initiated by activation of CD44‐associated phosphatase 2A (PP2A) and proceeds via ERK1/2 dephosphorylation without ERK1/2 degradation. Accordingly, CD44‐induced apoptosis could be mimicked by ERK1/2 inhibition, that also promotes EL4 cell apoptosis through the mitochondrial pathway. Thus, during haematopoietic stem cell reconstitution care should be taken not to interfere by a blockade of CD44 with haematopoiesis, which could be circumvented by selectively targeting leukemic CD44 isoforms. Beyond homing/settlement in the bone marrow niche, anti‐CD44 drives leukemic T cells into apoptosis via the mitochondrial death pathway by CD44 associating with PP2A. Uncovering this new pathway of CD44‐induced leukemic cell death provides new options of therapeutic interference.