Role of Exogenous Hyaluronan in Cancer Cell Signaling

Metastatic cancer is the leading cause of cancer mortality. Impeding cancer metastasis is a useful therapeutic tool for treating cancer. Metastasis occurs when tumor cells detach from the primary site, then migrate to a secondary tumor site. The extracellular matrix (ECM) is modified in the tumor environment and serves as both physical support and cell signaling regulator. The ECM is composed of insoluble proteins such as collagen and glycosaminoglycan hyaluronan. Hyaluronan is highly concentrated in tissues such as joints, skin and vitreous of eyes. The varying average molecular weight of HA is significant to its function; low molecular weight hyaluronan causes an inflammatory response whereas cells maintain a quiescent state in the presence of high molecular weight hyaluronan. Cells express multiple cell-surface receptors for hyaluronan. One of these receptors, layilin, has recently been identified as being involved in cell migration. Its interaction with hyaluronan is similar to that of CD44, the most prominent hyaluronan receptor. Layilin and CD44 interact with Src, a tumor promoting tyrosine kinase, and Merlin, a tumor suppressor. The phosphorylated forms of Src and Merlin stimulate their activities. Src, an oncogene, phosphorylates particular tyrosine residues in other proteins. High levels of Src phosphorylation are linked to cancer progression by activating signaling pathways. Merlin (Schwannomin) is a tumor suppressor and cytoskeletal- linker protein. When Merlin is phosphorylated, it acts as a tumor suppressor. However, loss of phosphorylation leads to aberrant tumor growth. Unregulated Merlin is associated with development of neutrofibromatosis, an autosomal, inherited disorder characterized by nervous system tumors. Merlin has an effect on cell motility by reducing phosphorylation of FAK, which contributes to cell migration and invasion.

In this study, HT1080 fibrosarcoma cells are used to investigate the effects of layilin and CD44 on cell proliferation, migration, and morphology in the presence or absence of low molecular weight or high molecular weight hyaluronan. shRNA was used to generate cell lines with diminished expression of layilin or CD44. Decreased expression of layilin resulted in slower motility compared to control cells. Western Blot analysis was performed to determine protein expression of layilin and CD44 knockdowns as well as different phosphorylation levels of Merlin and Src. Cell proliferation assays were studied to assess influence of layilin deficiency on growth. These results suggest that layilin plays a role in multiple aspects of cell behavior.