HA/CD44 Interaction with Cytoskeletal Proteins, Ankyrin, and ERM. Part 2
Hyaluronan-stimulated intracellular Ca2+ mobilization mediates important components of the CD44 signaling pathways. Some of these Ca2+-mediated pathways may be mediated by the Ca2+ binding protein calmodulin. It is known that calmodulin is involved in the activation of several important enzymes, including calcium/calmodulin-dependent protein kinase type II (CaMKII), a ubiquitous serine/threonine protein kinase. In HNSCC cells, CaMKII activation by HA/CD44-mediated Ca2+ mobilization results in the phosphorylation of diverse substrates that promote various cell functions, including motility, cell cycle progression, and proliferation. CaMKII phosphorylates the cytoskeletal protein, filamin. These HA/CD44-mediated effects on CaMKII and filamin lead to cytoskeleton reorganization and promote tumor cell migration. Wang et al49 linked HA/CD44-dependent CaMKII activity to topoisomerase II regulation in HNSCC cells. Topoisomerase II is a critical regulator of DNA topology and function. Hyaluronan treatment promoted CaMKII-dependent topoisomerase II phosphorylation, resulting in enhancement of topoisomerase II activity and decreased cytotoxicity of etoposide (a topoisomerase II poison).These HA/CD44-mediated effects on CaMKII and topoisomerase II activity enhanced tumor cell survival.
Another important effector of RhoA pathway signaling is Rho kinase (ROK). Activated ROK is known to phosphorylate a number of cytoskeletal proteins, such as myosin phosphatase and adducin, that are highly involved in tumor migration and to promote the secretion of MMPs involved in tumor invasion. Torre et al recently showed that HA/CD44 interaction increased ROK activity in HNSCC cells. Hyaluronan also promoted Rho kinase-mediated myosin phosphatase phosphorylation, resulting in enhanced tumor cell migration, and it increased activated MMP-2 and MMP-9 secretion.
RhoA/Ca2+ Signaling-Regulated Chemoresistance
HA/CD44 interaction has been shown to promote resistance to multiple chemotherapeutic agents in HNSCC, including cisplatin, methotrexate, doxorubicin (Adriamycin), and etoposide.38, 40, 49 Several cell signaling mechanisms appear to promote CD44-mediated chemoresistance in HNSCC, including EGFR-related signaling pathways (as already described here).38 Recent work by our groupsuggests that regulation of Ca2+ may also affect chemoresistance (unpublished data). The median inhibitory concentration IC50 for the chemotherapy agent methotrexate in the HNSCC cell line SCC4 is dependent on both the Ca2+ level and the presence of HA. SCC4 cells grown in 1.2 mmol/L Ca2+ medium had greater resistance to methotrexate than cells grown in low-Ca2+ medium, and the IC50 was increased in the presence of HA at both Ca2+ concentrations.
Phospholipase C and RhoA signaling, which mediate intracellular Ca2+ levels, has been shown to play roles in mediating chemoresistance in HNSCC. Wang et al demonstrated that HA-mediated cisplatin, methotrexate, and doxorubicin resistance could be eliminated with inhibition of PLC. Torre et al found that combined ROK and PI-3 kinase inhibition resulted in a synergistic prosurvival effect in the presence of cisplatin. Thus, HA-mediated chemoresistance in HNSCC may involve multiple pathways, including RhoA-mediated Ca2+ signaling.