Chaperome heterogeneity and its implications for cancer study and treatment

Abstract

The chaperome is the collection of proteins in the cell that carry out molecular chaperoning functions. Changes in the interaction strength between chaperome proteins lead to an assembly that is functionally and structurally distinct from each constituent member. In this review, we discuss the epichaperome, the cellular network that forms when the chaperome components of distinct chaperome machineries come together as stable, functionally integrated, multimeric complexes. In tumors, maintenance of the epichaperome network is vital for tumor survival, rendering them vulnerable to therapeutic interventions that target critical epichaperome network components. We discuss how the epichaperome empowers an approach for precision medicine cancer trials where a new target, biomarker, and relevant drug candidates can be correlated and integrated. We introduce chemical biology methods to investigate the heterogeneity of the chaperome in a given cellular context. Lastly, we discuss how ligand-protein binding kinetics are more appropriate than equilibrium binding parameters to characterize and unravel chaperome targeting in cancer and to gauge the selectivity of ligands for specific tumor-associated chaperome pools.

Keywords: 70 kilodalton heat shock protein (Hsp70); biomarker; cancer biology; cancer therapy; chaperome; chaperone; chemical biology; chemical probes; epichaperome; heat shock protein 90 (Hsp90); protein networks; stress response.

Figure 1.

Proteome stress biochemically remodels the chaperome in cancer. a, dynamic and hierarchical interactions between the chaperone machineries govern normal cellular proteostasis. Here, different chaperone machineries give rise to a network, with partially overlapping functions and specificities. Cellular stress, such as induced by Myc hyperactivation, increases the connectivity between distinct chaperone machineries; this is executed by an increase in the interaction strength among chaperones, co-chaperones, and other factors. A functionally and structurally interconnected chaperome network, as opposed to that of individual chaperone machineries, is formed. Because the formation of the interconnected chaperome creates an entity that is distinct, both thermodynamically and functionally, from its constituent chaperome units, this network was coined the epichaperome. b, tumors with the interconnected chaperome, i.e. epichaperome expressors, were termed type 1, whereas those with partly overlapping chaperome machineries were named type 2. The biochemical signature of HSP90 when part of the epichaperome is exemplified in the MDA-MB-468 breast cancer cell homogenates. Conversely, HSP90 of type 2 tumors is exemplified in ASPC-1 cells. IB, immunoblot. c, in type 1 tumors, but not in type 2, HSC70 is an abundant component of stable HSP90 chaperome complexes; this HSC70 pool, but not others, is depleted by the PU-H71 bait, a binder of HSP90 when incorporated into the epichaperome. Adapted from Ref. .

Figure 2.

Functional integration of the chaperome in type 1 tumors. a, in type 1 tumors, epichaperome integrity is maintained, even after a drastic reduction in HSP90 levels, by increasing the total expression levels and the participation in the epichaperome of other key epichaperome components. In these conditions of low HSP90 but high epichaperome, the function of oncogenic protein networks (see EGFR levels) and cell viability remained unaffected. At an inflection point, and as HSP90 continues to decrease, epichaperome networks collapse, oncogenic proteins are depleted, and cell death ensues. b, in type 2 tumors, HSP90 depletion results in immediate client (EGFR) depletion, but no remodeling of the chaperome takes place (HSC70 shown). IB, immunoblot. c and d, in type 1 but not in type 2 tumors, siRNA directed to HSP110, a co-chaperone of HSP/C70, depletes the HSP90 client protein EGFR and results in cell death. e, summary schematic of individual chaperome member inhibition and its outcome in type 1 and 2 cancer cells. a–d are adapted from Ref. and show a representative result or a mean value, as appropriate.

Figure 3.

ATP-competitive HSP90 inhibitor subtypes. a, overlay of the indicated inhibitors and their segregation based on subpocket occupancy. Adapted from Ref. . b, protein–ligand interactions representative of the loop-in conformation ligands (left) and of the helical conformation ligands (right). Chemical structure of the ligands is shown below. Adapted from Ref. .

Figure 4.

Binding of PU-H71 to different HSP90 pools. a, biochemical characterization of HSP90 in MDA-MB-468 and ASPC-1 cancer cells. Both cell lines contain similar total HSP90 levels (evaluated by immunofluorescence and immunoblot). MDA-MB-468 but not ASPC-1 contains HSP90 incorporated into epichaperome networks (evaluated by native PAGE and IEF). b and c, methods to evaluate the dissociation reaction kinetics of a ligand from different cellular HSP90 pools. One way is to compete a fluorescent or radioligand ligand from its receptor with a large excess of unlabeled ligand. The fluorescent ligand dissociates at a rate determined by K_off and does not rebind, because the unlabeled ligand takes its place. b, another way is to dilute an equilibrium mixture of ligand (A), receptor (B), and ligand–receptor complex (AB) and observe the time course of the dissociation of AB to establish new equilibrium concentrations of A and B. Monitoring of in vivo PK is in essence a surrogate of such a method. Adapted from Ref. . d, kinetic binding provides the selectivity of PU-H71 for epichaperome over other HSP90 pools. Reaction coordinates for a one-step binding event are shown on the right side to clarify the nature of equilibrium and kinetic binding parameters.

Figure 5.

Clinical assays to detect the stress-modified chaperome in cancer patients. a, PET scan is used to detect epichaperome-expressing solid tumors in cancer patients; b, flow cytometry-based assays is used to detect the epichaperome in liquid tumors; and c, native protein separation and analysis by isoelectric focusing followed by immunoblotting with native cognate antibodies are used for minute biopsy specimens. For the PET scan, the patient receives a minute dose of ¹²⁴I-labeled PU-H71 and then undergoes PET imaging to determine epichaperome expression (or the lack of) in the tumor. HSP90, involved in dynamic housekeeping chaperome complexes, although abundant and expressed essentially in all cells in the body, does not image in the scan. For the flow cytometry assay, the peripheral blood or bone marrow sample is stained with PU-FITC, and the single-cell signal (i.e. epichaperome expression or the lack of) is detected in a flow cytometer upon sorting of cell populations. Here, for example, blasts (the malignant population in a leukemia patient) are detected to be high epichaperome expressors. Conversely, normal cells (lymphocytes in the same leukemia patient), while high HSP90 expressors, do not stain with PU-FITC. For the biopsy, the frozen biospecimen is applied for IEF-IB analysis by the NanoPro IEF platform.

Figure 6.

Chemical biology approach for the investigation of the epichaperome. A combination of chemical biology probes, functional and biochemical assays applied to cells in their native environment, and complemented with classical biology approaches can be used to investigate epichaperome biology in cancer.