Cellular radiosensitivity and endocytosis: The role of PINCH1 and caveolin-1

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In normal and cancer cells, endocytosis plays a fundamental role in various cell functions such as survival, proliferation, metabolism, migration and signal transduction. Caveolae, predominantly formed by caveolins and cavins, mediate receptor-independent endocytosis and are essentially involved in the transfer of biochemical cues along inter– and intracellular signaling pathways. These actions fundamentally participate in the regulation of various cell functions, including the rapid responses to different types of stress. Our previous studies show that caveolin-1 is over-expressed in malignant tumors, serves as a critical component for proper integrin signaling and acts as a promising cancer target as its downregulation sensitizes human cancer cells to radiochemotherapy. As caveolin-1 coalesces and co-precipitates with cytoskeletal proteins and focal adhesion proteins - like the adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1), which, itself, is a key determinant of radio- and chemoresistance - we investigated this interaction in the context of endocytosis in the ongoing grant proposal. Our preliminary data show that (i) the expression levels of both caveolae associated proteins, caveolin-1 and cavin1, are PINCH1-dependent and (ii) PINCH1 critically regulates caveolae motility upon X–ray irradiation through interactions with caveolin–1 and cavin1. Given the paucity of information about radiation-modulated endocytosis, we here, in this follow-up grant proposal, describe further in-depth investigations on (i) determining the interaction dynamics of PINCH1–cavin1 and PINCH1–caveolin–1 upon X–ray irradiation, (ii) the identification of the physical binding sites for this interaction on PINCH1, cavin1 and caveolin–1, (iii) uncovering the PINCH1–dependent organization of actin and tubulin networks and how critical these are for the motility of cavin1 and caveolin–1 upon X–ray irradiation, and (iv) identifying the molecular recruitment mechanisms of cavin1 and caveolin–1 by PINCH1 under radiogenic stress. The proposed follow-up project addresses these questions by means of mouse embryonic PINCH1-/- fibroblasts reconstituted with different variants of PINCH1 plus co-transfection with wildtype and mutated forms of caveolin-1 and cavin1. Through the visualization of their interaction dynamics and the identification of the physical binding sites between PINCH1, cavin1 and caveolin–1, this follow–up project aims to provide in–depth understanding of how caveolae formation and function are regulated, how these proteins contribute to resistance processes and associated inter– / intracellular signaling networks that may be exploitable for enhancing radiochemosensitivity through targeting of particular components of caveolae associated endocytosis.