Copolymers with hyalurinan branches as efficient antagonist of CD44 signalling in breast cancer cells

Final FoReCaST Conference “2D and 3D in vitro Tissue Models for Drug Screening: Strengths and Limitations of the Cancer Models”, 27-29 October 2021, Porto, PortugalCD44 is the main hyaluronan (HA) receptor involved in the communication of cells with their microenvironment. CD44/HA interactions and following signaling cascade(s) depend on HA size. In cancer, HA with high molecular weight interacts, organizes, and clusters several extracellular and membrane protei ns, including CD44, to form a dense and functional pericellular coat. [1] These interactions activate anti-apoptotic, pro-invasion, and migration signaling pathways, promote drug resistance, facilitate intravasation, protect against anoikis during circulation, and contribute to ectopic tissue colonization. [2] Inhibition of such CD44/HA interactions and CD44 clustering has been thus explored as a therapeutic target. [3] In this work, we synthesized poly(hydroxyethyl)methacrylate graft HA (HEMA-g-HA) by oxime reaction at the reducing end of HA (4.8 kDa). We studied the interactions of these copolymers with CD44 by Surface Plasmon Resonance (SPR) and compared them against HA of low (4.8 kDa) and high (1.35 MDa) molecular weight. All copolymers bind to CD44 with the high association and dissociation constants: we observed a much higher affinity of CD44 to HEMA-g-HA than to HA (4.8 kDa) and similar to the measured for HA (1.35 MDa). On the other hand, the degradation of HEMA-g-HA by hyaluronidase is slower than free HA as shown by isothermal titration calorimetry (ITC). These results indicated that HEMA-g-HA could be a good antagonist of CD44/HA signaling in cancers. To check this hypothesis, we studied the binding of HEMA-g-HA-Rho to GFP-CD44 in transfected Sk-Br-3 cells by Forster resonance energy transfer (FRET) microscopy. FRET showed no significant difference between the copolymer, HA of 4.8 kDa, and 1.35 MDa HA, suggesting the same binding sites. Competitive binding assays demonstrated similar activity for HEMA-g-HA and HA of 4.8 kDa, but Western blot and immunocytochemistry analysis evidenced that the copolymer regulates CD44 expression and location, promoting receptor declustering and translocation to the cytoplasm - alternations that ultimately lead to cell deathThe authors thank the Portuguese Foundation for Science and Technology (FCT) (SFRH/BD/ 114847/2016, PTDC/QUI-POL/28117/2017, CEECIND/00814/2017)N