The Converging Roles of Cholesterol and C-Terminal Src Kinase in the Regulation of Extracellular Matrix Degradation at Invadopodia

Bicanova, Kristyna (2014). The Converging Roles of Cholesterol and C-Terminal Src Kinase in the Regulation of Extracellular Matrix Degradation at Invadopodia. PhD thesis The Open University.



Metastasis, the leading cause of mortality in cancer patients, is the dissemination of cancer cells from the primary tumour and spread to the distant sites of the body. This is a complex process during which tumour cells need to overcome several natural barriers to gain entry into the bloodstream and thus allow formation of secondary tumour in distant location. In the last two decades, much effort has been focused on showing that tumour cells use specialized actin-based membrane protrusions termed invadopodia to perform matrix degradation. Invadopodia gain their protrusive capacity combining the mechanical force of actin polymerization with the chemical activity of matrix degradation. As such, invadopodia are F-actin-rich structures enriched in integrins, tyrosine kinases signalling machinery, soluble and membrane proteases, including matrix metalloproteases (MMPs), and actin-associated proteins. How all these components are specifically recruited to the ECM degradation sites has not been fully clarified yet.

An emerging model describes invadopodia as dynamic cellular platforms where the signalling, membrane trafficking and cytoskeleton remodelling converge upstream of ECM degradation at spatially confined cholesterol-rich membrane compartments. Despite the field of invadopodia biogenesis and function is still a very recent, it is witnessing an increasing interest and an increasing number of molecular players have been identified in the last two decades. Although the existence of the invadopodia-like structures in vivo settings still needs to be determined, invadopodia represent powerful experimental paradigm to study the tight integration between the signalling, the membrane trafficking and cytoskeleton remodelling upstream of ECM degradation, the rate-limiting step in cell invasion, and might provide better understanding of cancer cell invasion and metastasis.

In order to investigate the cholesterol-rich lipid raft feature of invadopodia, I followed three lines. First, I showed that inhibition of cholesterol formation at penultimate step of its biosynthesis, and subsequent accumulation of desmosterol, blocks formation and function of invadopodia, thus demonstrating that the central role of cholesterol is connected to its presence in functional lipid rafts. In the second approach, I found that the SFKs inhibitory kinase Csk is a negative regulator of invadopodia-mediated ECM degradation and its role depends on the localization in the cholesterol-rich lipid rafts. Finally, I demonstrated that free cholesterol-dependent ARF6-associated recycling pathway might be involved in the trafficking to invadopodia, while the ARF6-pathway constituents are localized at ECM degradation sites and ARF6-specific cargo CD 147 is recycled to invadopodia.

Taken together, my findings provide novel insight towards the elucidation of invadopodia as specialized cholesterol-dependent membrane domains where signal transduction and membrane trafficking events might be temporally and spatially confined.

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