Evolutionarily conserved dual lysine motif determines the non-chaperone function of secreted Hsp90alpha in tumour progression

Both intracellular and extracellular heat shock protein-90 (Hsp90) family proteins (alpha and beta) have been shown to support tumour progression. The tumour-supporting activity of the intracellular Hsp90 is attributed to their N-terminal ATPase-driven chaperone function. What molecular entity determines the extracellular function of secreted Hsp90 and the distinction between Hsp90 alpha and Hsp90 beta was unclear. Here we demonstrate that CRISPR/Case9 knocking out Hsp90 alpha nullifies tumour cells' ability to migrate, invade and metastasize without affecting the cell survival and growth. Knocking out Hsp90 beta leads to tumour cell death. Extracellular supplementation with recombinant Hsp90 alpha, but not Hsp90 alpha, protein recovers tumourigenicity of the Hsp90 alpha-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in the Hsp90 alpha subfamily that determine the extracellular Hsp90 alpha function. Hsp90 beta subfamily lacks the dual lysine motif and the extracellular function. Substitutions of gly-262 and thr-269 in Hsp90 beta with lysines convert Hsp90 beta to a Hsp90 alpha-like protein. Newly constructed monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90 alpha inhibits both de novo tumour formation and expansion of already formed tumours in mice. This study suggests an alternative therapeutic approach to target Hsp90 in cancer, that is, the tumour-secreted Hsp90 alpha, instead of the intracellular Hsp90 alpha and Hsp90 beta.