Role of CD44-cytoskeleton interaction in the regulation of prostate tumor cell activation

CD44 is a glycosylated adhesion molecule which may undergo alternative splicing to generate variant isoforms. A number of CD44 variant isoforms expressed by tumor cells have been correlated with metastatic and proliferative behavior. In this study we have characterized CD44 isoform expression on three prostate cancer cell lines, ALVA-31, PPC-1, and LNCaP. We have found that ALVA-31 and PPC-1 cells displaying tumorigenesis and invasiveness in nude mice express multiple CD44 isoforms, including CD44s or CD44v(ex14/v10). In contrast, LNCaP cells exhibiting a less malignant phenotype do not express any of the CD44 forms at the RNA or protein level. These results suggest a correlation between CD44 variant (CD44v) expression and aggressive prostate tumor behavior. Furthermore, stable transfection of CD44s and CD44v(ex14/v10) into LNCaP can promote LNCaP cell adhesion to HA and provide growth advantage to LNCaP cells. In addition, CD44s or CD44v(ex14/v10) significantly promote cell transformation and migration, which are key events associated with tumor progression.To study the role of CD44s-ankyrin interaction in regulating human prostate tumor cells, we have constructed several CD44s cytoplasmic deletion mutants and transfected them into LNCaP. Our results indicate that a critical region of 15 amino acids between aa304 and aa318 of CD44s is required for ankyrin binding. Deletion of this 15aa ankyrin-binding sequence from CD44s results in a drastic reduction of HA-mediated binding/cell adhesion, cell migration, Src p60 kinase(s) interaction and anchorage-independent growth in soft agar. In addition, we demonstrated that the N-terminal repeat domain in ankyrin is directly involved in binding to CD44 cytoplasmic domain using several recombinant proteins including CD44cyt-FLAG and AnkRD-GST.Taken together, all these findings have important implications in understanding how the binding of ankyrin to the cytoplasmic domain of CD44 plays a pivotal role in regulating HA-mediated functions as well as Src kinase activity and prostate tumor cell activation