Abstract 1268: Externalized cytokeratin 8 : A relevant target at the crossroads of microenvironment and intracellular signaling

Abstract

Abstract Accumulating evidence suggests the unusual presence of proteins at the external leaflet of plasma membrane of cancer cells while, in normal cells, these proteins are expressed in the intracellular compartments. They lack transmembrane domains and signal sequences that allow delivery to the plasma membrane and their role is not fully understood. As such, aberrantly externalized proteins represent a promising source of targets for cancer therapy. In order to find new targets for the treatment of metastatic colorectal cancer (CRC), we performed a comparative dynamic proteomic analysis of Isreco-1, a cell line characterized by a high invasiveness capacity. Among a set of proteins associated with invasion, we identified CK8, an intermediate filament protein of epithelial cells cytoskeleton involved in the maintenance of cell integrity of normal glandular epithelial cells. CK8 was shown to be expressed both intracellularly and associated at the plasma membrane of highly invasive Isreco-1 line (KRAS mutant) as well as in two other cell lines i.e. HCT116 (KRAS mutant) and HT29 (KRAS wild-type), by immunofluorescence. Using a biochemical approach, we demonstrated in all 3 cell lines, that plasma membrane-associated CK8 was strongly anchored to the plasma membrane, and subsequently referred as externalized CK8 (eCK8). eCK8 was co-localized with plasminogen and urokinase-type plasminogen activator (uPA) at the plasma membrane of Isreco-1 cells, consistent with its previously reported role as a plasminogen receptor. Among several murine monoclonal antibodies (MAbs) targeting specifically eCK8, one was able to reduce plasminogen induced invasion in vitro (Isreco-1) as well as tumor growth in vivo (Isreco-1 and HCT-116 subcutaneous xenograft models). It was specific of an extra-cellular portion of eCK8. Besides the inhibition of tumor growth, tumors were strikingly necrotic and characterized by apoptosis activation. No difference in terms of proliferation and microvascular density was observed. Altogether, our results suggest that in colorectal cancer, CK8 may be externalized and strongly anchored to the plasma membrane in highly invasive cells. eCK8 co-localize with plasminogen and uPA. Targeting a specific portion of eCK8 was associated with decreased tumor growth and apoptosis activation. We suggest that this particular domain of eCK8 is essential for bidirectional signaling, toward the tumor cell microenvironment by favouring the invasion process and the intracellular compartment by inhibiting apoptosis. Because the combination of cell invasion and inhibition of apoptosis are key mechanisms involved during metastasis, externalization of CK8 may represent a key event of the metastatic process and a relevant target in CRC, including in tumors with KRAS mutation. Citation Format: Marie Alexandra Albaret, Claudine Vermot-Desroches, Arnaud Paré, Jean-Xavier Roca-Martinez, Jad Essely, Laetitia Gerossier, Johan Brière, Nathalie Pion, Lucie Malet, Boris Vuillermoz, Carine Rousset, Hichem-Claude Mertani, Pierre Saintigny, Jean-Jacques Diaz. Externalized cytokeratin 8 : A relevant target at the crossroads of microenvironment and intracellular signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1268.