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Tumor dissemination to distant site and seeding of metastasis is the major clinical problem in cancer. We aimed to understand the molecular underlying mechanisms through study endocytic networks, which by controlling key steps of intracellular trafficking pathways have a pervasive role in decoding spatial information and signalling, influencing tumor initiation, self renewal and cell invasive plasticity. Our central hypothesis was that the identification and characterization of the mechanisms of action of critical membrane trafficking proteins that are deregulated during cancer initiation and progression may be exploited to identify new prognostic markers and design novel cancer therapies as well as shed light on the unexpected consequences of fundamental cellular pathways. We further aimed at elucidating whether pervasive control of membrane trafficking network may impinge on supra-cellular biochemical and biomechanical properties.
A number of recent studies have revealed that the processes through which tumor disseminate are extremely diverse, flexible and adaptable, leading to the notion of plasticity in the modes of cell migration and invasion. This plasticity is instrumental to ensure that cancer cells successfully deal with intricate and diverse extracellular micro-environments. These adaptive responses also provide migratory escape strategies after pharmacotherapeutic intervention, by prompting alternative mechanisms of cancer cell dissemination that overcome single-pathway-hitting pharmacological weapons. Understanding of the molecular determinants at the basis of the plasticity of tumor invasion appears therefore essential to define novel rational strategy for the development of pharmacological weapons to combat tumor dissemination.
The critical pathways and cellular processes underlying the plasticity of tumor cell motility have only begun to be identified. We focused on a set of pivotal trafficking hubs known to dramatically perturb virtually all endocytic network and further search for selected components of the endomatrix that may specifically impact on cell migratory and invasive processes.
Within this context we found that:
-RAB5, a master regulator of endocytic internalization, has pervasive effects on the mode of individual cell migration not only by controlling the extent of actin based migratory and mesenchymal protrusion, but also by promoting pericellular proteolytic activities essential to enable cancer cells to navigate into dense, highly desmoplastic tissues. This is achieved in conjunction with another RAB family members, RAB4A, that specifically controls the fast recycling routes of membrane-bound metalloproteases. Not surprisingly the RAB5/RAB4A axis is hijacked by human breast cancer and its elevation predicts poor outcome. This pathway also operates in vivo by controlling the conversion from locally growing in situ ductal breast carcinoma to invasive carcinoma. Remarkably, a different harm of the RAB5 pathways centered on its regulatory proteins RN-tre (a GTPase activating molecule) also controls the ability of cells to migrate chemotactically on adherent substrates by promoting the turnover of integrin receptors.
More recently, we were able to show that RAB5A has major impact also on the regulation of collective cells motility, the preferred mode of migration utilized by virtually all cancer cells of mesenchymal and epithelial origin. We could show that RAB5A global effects on endocytosis are essential to control the biomechanical properties of cell ensembles and enhance motility by promoting a fluidization of cell monolayers, that literally start flowing like fluids upon perturbation of this gene.
-Through candidate and unbiased approaches we identified novel key endocytic molecules impacting on different aspect of the migratory strategies adopted by breast cancers. Briefly, we showed that the F-bar containing membrane deforming protein-interactor of CDC42-CIP4, is critical in controlling the epithelial to mesenchymal morphological conversion by regulating junctional strength and the dynamics and turnover of junctional molecules. Remarkably, CIP4 is elevated in a subset of breast and lung carcinoma and predict poor outcome.
-Along similar line, through two RNAi-based imaging screening we identified RAB2A as pivotal in promoting a matrix degradative program of cell invasion in vitro as well in vivo. RAB2A, a Golgi localized protein unexpectedly acts on post endosomal trafficking and facilitates the delivery of membrane bound proteases as well as control the distribution of E-cadherin.
The above-mentioned results represent only few examples of the novel set of finding obtained within the Metaendomatrix project that were key to support the notion that endocytic factors are pivotal in providing cells with alternative mechanisms and strategies of migrations and invasion in complex environments.
In summary, our results provide important contribution toward the understanding of the molecular underpinning and wiring onto which each of our cells is built. They further help in defining the molecular basis of cancer by delineating membrane trafficking-dependent mechanisms of cancer migration, invasion and metastasis, ultimately paving the way for the development of radically novel cancer therapies.