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 (α and ß) 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 Hsp90α and Hsp90ß was unclear. Here we demonstrate that CRISPR/Case9 knocking out Hsp90α nullifies tumour cells' ability to migrate, invade and metastasize without affecting the cell survival and growth. Knocking out Hsp90ß leads to tumour cell death. Extracellular supplementation with recombinant Hsp90α, but not Hsp90ß, protein recovers tumourigenicity of the Hsp90α-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in the Hsp90α subfamily that determine the extracellular Hsp90α function. Hsp90ß subfamily lacks the dual lysine motif and the extracellular function. Substitutions of gly-262 and thr-269 in Hsp90ß with lysines convert Hsp90ß to a Hsp90α-like protein. Newly constructed monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90α 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 Hsp90α, instead of the intracellular Hsp90α and Hsp90ß.

Modulation of type VII collagen (anchoring fibril) expression by retinoids in human skin cells.

We examined the effects of retinoids on the expression of type VII collagen, a major component of anchoring fibrils, in human keratinocytes and amnion cells (WISH). All-trans retinoic acid (RA) (5 X l0(-6) M) decreased the steady-state levels of type VII collagen mRNA by at least 80% after 18 h. The inhibition was evident within 6 h after the addition of RA, maximal at 18 h, and was dose-dependent. Reduction of type VII mRNA expression also occurred when cell cultures were incubated with retinol, retinal, and 13-cis RA. Retinoid-mediated inhibition of type VII collagen mRNA expression was observed in keratinocytes growing in either serum-free keratinocyte growth medium (KGM) or KGM supplemented with 1.4 mM Ca2+. Cycloheximide blocked RA-mediated inhibition of type VII collagen mRNA, demonstrating the need for de novo protein synthesis. The mRNA levels for fibronectin and glyceraldehyde phosphate dehydrogenase were not affected by the retinoids, suggesting selective inhibition on type VII collagen expression. In addition, the decrease in type VII collagen mRNA was accompanied by a parallel decrease in secretion of the 290 kDa, type VII collagen alpha chains.