CD44 in cancer progression: adhesion, migration and growth regulation.

It is well established that the large array of functions that a tumour cell has to fulfil to settle as a metastasis in a distant organ requires cooperative activities between the tumour and the surrounding tissue and that several classes of molecules are involved, such as cell-cell and cell-matrix adhesion molecules and matrix degrading enzymes, to name only a few. Furthermore, metastasis formation requires concerted activities between tumour cells and surrounding cells as well as matrix elements and possibly concerted activities between individual molecules of the tumour cell itself. Adhesion molecules have originally been thought to be essential for the formation of multicellular organisms and to tether cells to the extracellular matrix or to neighbouring cells. CD44 transmembrane glycoproteins belong to the families of adhesion molecules and have originally been described to mediate lymphocyte homing to peripheral lymphoid tissues. It was soon recognized that the molecules, under selective conditions, may suffice to initiate metastatic spread of tumour cells. The question remained as to how a single adhesion molecule can fulfil that task. This review outlines that adhesion is by no means a passive task. Rather, ligand binding, as exemplified for CD44 and other similar adhesion molecules, initiates a cascade of events that can be started by adherence to the extracellular matrix. This leads to activation of the molecule itself, binding to additional ligands, such as growth factors and matrix degrading enzymes, complex formation with additional transmembrane molecules and association with cytoskeletal elements and signal transducing molecules. Thus, through the interplay of CD44 with its ligands and associating molecules CD44 modulates adhesiveness, motility, matrix degradation, proliferation and cell survival, features that together may well allow a tumour cell to proceed through all steps of the metastatic cascade.

Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells.

T cell activation is accompanied by actin-mediated receptor clustering and reorganization of lipid rafts. It has been suggested that costimulatory molecules might be involved in these processes. We here provide evidence that engagement of the adhesion molecule CD44 initiates cytoskeletal rearrangement and membrane reorganization in T cells. Cross-linking of CD44 on a T helper line was accompanied by adhesion, spreading and actin bundle formation. These processes were energy dependent and required an intact actin and microtubule system. They involved the small GTPase Rac as evidenced by the absence of spreading in cells overexpressing a dominant negative form of Rac. The CD44 initiated reorganization of the cytoskeleton was associated with the recruitment of CD44 and the associated tyrosine phosphokinases p56(lck) and p59(fyn) into glycolipid enriched membrane microdomains (GEM). We interpret the data in the sense that CD44 functions as a costimulatory molecule in T cell activation by inducing actin cytoskeletal rearrangements and membrane protein and lipid reorganization including its association with GEMs. Due to the association of CD44 with lck and fyn this colocalization with the TCR allows an abundant provision of these kinases, which are essential to initiate the TCR signaling cascade.

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.

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 Ick 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.

Regulation of phospholipid biosynthesis by Ca(2+)-calmodulin-dependent protein kinase inhibitors.

Inhibitors of Ca(2+)-calmodulin (CaM)-dependent protein kinases strongly modify phospholipid metabolism. Two compounds, KN62 and KT5926 recognized as blockers of Ca(2+)-CaM-dependent protein kinase II, induced a specific increase in phosphatidylserine (PtdSer) synthesis without noticeable changes in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) biosynthesis. The increase of PtdSer synthesis was dependent on the presence of Ca2+ in the incubation medium and was impaired in cells whose Ca2+ stores were depleted by pretreatment with CD3 mAb, thapsigargin or EGTA. The mechanism of the stimulation of PtdSer synthesis by these two compounds seems to involve an accumulation of Ca2+ into the endoplasmic reticulum, possibly due to an increased activity of the endoplasmic reticulum Ca(2+)-ATPase. By contrast, ML-7 and ML-9, two inhibitors of the myosin light chain kinase (MLCK), another Ca(2+)-CaM-dependent kinase, were both capable of increasing PtdSer synthesis and decreasing PtdCho and PtdEtn synthesis, reproducing the effect previously described with CaM-antagonists. The increase of PtdSer caused by ML-7 and ML-9 was Ca(2+)-dependent while the inhibition of PtdCho and PtdEtn synthesis was not. The use of these four protein kinase inhibitors thus suggests the possible existence of two CaM-dependent pathways that differentially regulates phospholipid metabolism in T cells.

The protein tyrosine kinase p56(lck) regulates the serine-base exchange activity in Jurkat T cells.

Different classes of protein kinase inhibitors for protein kinase C, cAMP-dependent protein kinase or protein tyrosine kinases have been studied for their effect on phospholipid metabolism. The results show that among the compounds studied, only 4'-aminohydroxyflavone (AHF), previously described as a specific inhibitor of the protein tyrosine kinase p56(lck), markedly increased phosphatidylserine synthesis in Jurkat T cells. The biosyntheses of phosphatidylcholine and phosphatidylethanolamine were not affected. Also, the synthesis of phospholipids from tritium-labeled fatty acid as precursor was left unchanged by the p56(lck) inhibitor. The decreased phosphatidylserine synthesis induced when triggering the CD3-TCR complex was impaired by AHF, suggesting that p56(lck) could be implicated in the regulation of the serine-base exchange enzyme system. Direct evidence for the participation of p56(lck) in the regulation of the serine-base exchange enzyme system was obtained by using p56(lck)-deficient Jurkat cells (J.CaM 1.6) in which the basal base exchange activity was markedly increased and on the other hand AHF had no effect. In addition, transfection of J.Cam 1.6 cells with p56(lck)-cDNA allowed recovery of the AHF activity.

Tyrphostin A9 inhibits calcium release-dependent phosphorylations and calcium entry via calcium release-activated channel in Jurkat T cells.

The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.

Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat cells.

The calcium release-activated channel (CRAC) opened in Jurkat cells activated either with CD3 monoclonal antibody or the endoplasmic reticulum Ca2(+)-ATPase blocker, thapsigargin, is blocked by La3+ with an IC50 of 20 nM. Similarly, the entry of Mn2+, used as a surrogate for Ca2+, is also blocked by submicromolar La3+ concentrations. La3+ seems to play its role simply by plugging the CRAC because this ion does not penetrate the cells, as demonstrated by chelation experiments with EGTA. Blocking the Ca2+ influx in activated Jurkat cells results in a lack of expression of CD25, a chain of the interleukin-2 receptor and of CD69, a marker of T-cell activation. By contrast, the very early steps of the T-cell signalling pathway such as the release of Ca2+ from intracellular stores and the subsequent inhibition of phosphatidylserine synthesis are not affected by La3+.