Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.

OBJECTIVE: Invasive synovial fibroblasts are suggested to be the major effectors of cartilage and bone destruction, and this aggressive phenotype can lead to irreversible damage. In cancer cells, invasion across tissue boundaries and metastasis have recently been shown to depend on the capacity of the cells to breach the basement membrane, a process that was linked to the formation of the actin-rich cell protrusions called invadopodia. This study was undertaken to investigate whether arthritic synovial cells use invadopodia to invade and degrade cartilage components. METHODS: Fibroblast-like synoviocytes (FLS) from control rats or rats with collagen-induced arthritis (CIA) were cultured on fluorescent matrix in the presence of Src inhibitors or were transfected with wild-type or variants of Src kinases. The in vivo effect of Src inhibition on cartilage degradation and invasion was studied in a rat model of CIA. RESULTS: FLS from rats with CIA produced more invadopodia-like structures than did FLS from control rats, leading to increased extracellular matrix degradation. Furthermore, c-Src activation was increased in synovial cells from rats with CIA, and Src activity was found to mediate the formation of invadopodia. Pharmacologic blockade of Src activity by PP2 or intraarticular expression of a c-Src-specific short hairpin RNA in the CIA model reduced synovial membrane hyperplasia and cartilage degradation, an event linked to decreased invadopodia formation by synovial fibroblasts. CONCLUSION: This study demonstrates that inhibition of invadopodia formation in arthritic synovial cells leads to a direct effect on extracellular matrix degradation in vitro and in vivo, making invadopodia a relevant therapeutic target for interfering with this process.

Multiple alternative splicing markers for ovarian cancer.

Intense efforts are currently being directed toward profiling gene expression in the hope of developing better cancer markers and identifying potential drug targets. Here, we present a sensitive new approach for the identification of cancer signatures based on direct high-throughput reverse transcription-PCR validation of alternative splicing events. This layered and integrated system for splicing annotation (LISA) fills a gap between high-throughput microarray studies and high-sensitivity individual gene investigations, and was created to monitor the splicing of 600 cancer-associated genes in 25 normal and 21 serous ovarian cancer tissues. Out of >4,700 alternative splicing events screened, the LISA identified 48 events that were significantly associated with serous ovarian tumor tissues. In a further screen directed at 39 ovarian tissues containing cancer pathologies of various origins, our ovarian cancer splicing signature successfully distinguished all normal tissues from cancer. High-volume identification of cancer-associated splice forms by the LISA paves the way for the use of alternative splicing profiling to diagnose subtypes of cancer.