Role of CD44 and its interaction with CXCR4 in the Pathogenesis of Chronic Lymphocytic Leukemia

The processes that characterize tumor progression, like survival and growth of the primary tumor, invasiveness, migration and settlement of metastatic cells, are strongly connected with the interaction between components of the microenvironment, like components of the extracellular matrix (ECM) and signaling, molecules and cancer cells. The glycosaminoglycan Hyaluronan (HA) is one of the most importants components of the ECM, and induces intracellular signaling through its interaction with CD44, its main receptor. This interaction results in proliferation and migration signals. Smaller Hyaluronan fragments (sHA) can be produced from high molecular weight Hyaluronan (hHA), which can be digested by a class of enzymes called Hyaluronidases. This can happen during pathological processes like inflammation or tumor progression. HA of different lenghts produce different (or even opposite) effects on cells. Chemokines are other important components of the extracellular environment. On of these is CXCL12, which, through its receptor CXCR4, provides chemotactic and migration signals. There are evidence that suggest that CXCR4 and CD44 can interact, possibly by the formation of complexes of receptors. The migration of Hematopoietic Stem Cells (HSCs) during the homing process, guided by CXCL12, can be blocked by an antibody that prevents the binding of CD44 to HA. In the same way, the migration of leukemic stem cells in pathologies like Acute Myeloid Leukemia (AML), which is dependent on CD44, can be blocked by antibodies against CXCR4. In my master thesis I investigated the effect of different forms of HA and of an antibody against CD44 on the signalling cascade of CXCL12/CXCR4 axis in Mec1, a Chronic Lymphocytic Leukemia (CLL) cell line, and in CLL cells from patient material. I cultured Mec1 and HepG2Iso (epatic carcinoma cell line, used as positive control for some experiments) and performed some induction experiment using CXCL12 alongside HA. By performing western blot analysis I showed that while hHA enhanced the effect of CXCL12-induced phosphorylation of Erk (a component of the MAPK cascade), sHA can reduce this activation. Both effects appear to be dose-dependent. Also, the 3 treatment with BU52 (an antibody that inhibits the binding of CD44 and HA) reduces the increased effect of hHA on CXCL12-induced Erk phosphorylation in Mec1. Also, I performed Boyden Chamber Migration assays to show that the migration of Mec1 cells to a CXCL12 gradient is partially blocked by the pre- treatment with sHA. The data I collected suggested that the interaction between CXCR4 and CD44 can have an important role in the pathogenesis of CLL, in processes like tumor progression or migration to the niche