Extracellular Heat Shock Protein (Hsp)70 and Hsp90α Assist in Matrix Metalloproteinase-2 Activation and Breast Cancer Cell Migration and Invasion

Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90α) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90α is unknown. Intracellular Hsp90α commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90α functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90α mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90α in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90α and MMP-2 in vitro. This co-chaperone complex enhances Hsp90α-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90α increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development

Reconstitution of a Functional Duck Hepatitis B Virus Replication Initiation Complex from Separate Reverse Transcriptase Domains Expressed in Escherichia coli

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (ɛ) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by ɛ and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of ɛ-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism

A biochemical rationale for the anticancer effects of Hsp90 inhibitors: Slow, tight binding inhibition by geldanamycin and its analogues

Heat shock protein (Hsp)90 is emerging as an important therapeutic target for the treatment of cancer. Two analogues of the Hsp90 inhibitor geldanamycin are currently in clinical trials. Geldanamycin (GA) and its analogues have been reported to bind purified Hsp90 with low micromolar potency, in stark contrast to their low nanomolar antiproliferative activity in cell culture and their potent antitumor activity in animal models. Several models have been proposed to account for the ≈100-fold-greater potency in cell culture, including that GA analogues bind with greater affinity to a five-protein Hsp90 complex than to Hsp90 alone. We have determined that GA and the fluorescent analogue BODIPY-GA (BDGA) both demonstrate slow, tight binding to purified Hsp90. BDGA, used to characterize the kinetics of ligand–Hsp90 interactions, was found to bind Hsp90α with k(off) = 2.5 × 10(−3) min(−1), t(1/2) = 4.6 h, and K(i)* = 10 nM. It was found that BDGA binds to a functional multiprotein Hsp90 complex with kinetics and affinity identical to that of Hsp90 alone. Also, BDGA binds to Hsp90 from multiple cell lysates in a time-dependent manner with similar kinetics. Therefore, our results indicate that the high potency of GA in cell culture and in vivo can be accounted for by its time-dependent, tight binding to Hsp90 alone. In the broader context, these studies highlight the essentiality of detailed biochemical characterization of drug–target interactions for the effective translation of in vitro pharmacology to cellular and in vivo efficacy