Hsp90 C-Terminal Inhibitors Exhibit Antimigratory Activity by Disrupting the Hsp90α/Aha1 Complex in PC3-MM2 Cells

Abstract

Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure–function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.

Endoplasmic Reticulum-resident Heat Shock Protein 90 (HSP90) Isoform Glucose-regulated Protein 94 (GRP94) Regulates Cell Polarity and Cancer Cell Migration by Affecting Intracellular Transport

“…Previously, we reported that HSP90␣ forms a complex with the late phase co-chaperone, AHA1, and that the HSP90␣-AHA1 complex is transported toward the leading edge of a migrating cell in secretory vesicles (Fig. 6A) (49). Our cell organelle fractionation studies also revealed that cytosolic HSP90␣ levels were dependent upon GRP94 (Fig.…”

“…3). To determine whether integrin molecules are dependent upon GRP94 or other HSP90 isoforms, GRP94, HSP90␣, and HSP90␤ knockdown cells were constructed (16,49). Integrin ␣L was included in the Western blotting analysis because it was previously reported to be dependent on GRP94 (11).…”

“…Localization of the HSP90␣-AHA1 complex in the secretory vesicle was determined by co-localization and co-immunoprecipitation with a secretory vesicle marker protein, RAB3GAP1 (Fig. 6A) (49). Strongly supporting the co-localization of HSP90␣, AHA1, and RAB3GAP1, image quantification showed that Pearson's co-efficient (r) was 0.92 Ϯ 0.009 (n ϭ 25, p Ͻ 0.001) for HSP90␣ and AHA1 and 0.95 Ϯ 0.003 (n ϭ 25, p Ͻ 0.001) for RAB3GAP1 and AHA1.…”

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation

“…As such, and due to its association with Hsp90, the presence of Aha1 in exosomes (Fig. 8) comes as no surprise since Hsp90-Aha1 complexes have been shown before to be secreted by human cells via exosomes (Ghosh et al 2015;Park et al 2010).…”

Diverging Novobiocin Anti-Cancer Activity from Neuroprotective Activity through Modification of the Amide Tail

“…22 Those studies indicated that the noviose sugar was responsible for binding Hsp90 while the benzamide side chain present in KU-174 ( Figure 1) interacted with Aha1, and, when combined, manifested anti-cancer activity. 22 In contrast, replacement of the benzamide with an acetamide chain, as in the case of KU-32, did not disrupt the Hsp90α/Aha1 complex and, consequently, did not exhibit anti-cancer activity. 22 In an effort to systematically investigate the differences manifested by the alkyl and aryl containing amide side chains, structure−function studies were investigated to identify the point of divergence at which a neuroprotective agent is transformed into an anti-cancer agent.…”