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Oncotarget. 2016 Mar 15;7(11):12975-96. doi: 10.18632/oncotarget.7349.

Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.

Author information

1
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
2
Urologic and Diagnostic Radiology, Yale School of Medicine, New Haven, CT 06520-8058, USA.
3
Urologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
4
Department of Chemistry and Physics, Augusta University, Augusta, GA 30912, USA.
5
Arno Therapeutics, Flemington, NJ 08822, USA.
6
Molecular and Translational Science, United States Medicinal Chemistry and Pharmacognosy, School of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
7
School of Biological Sciences and Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia.

Abstract

We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone - chaperone and chaperone - client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 -dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 -induced activation of ER stress signaling and maintained mTOR activity; AR-12 -mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.

KEYWORDS:

ATPase; OSU-03012; chaperones; pazopanib; sorafenib

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