We present herein a rapid and simple chemical-proteomics method for surveying tumor oncoproteins (Supplementary Fig. 8
). The method takes advantage of PU-H71’s ability to (i) preferentially bind to a pool of Hsp90 associated with oncogenic client proteins, and (ii) trap Hsp90 in an oncoclient-bound configuration. We propose that this approach provides a powerful tool to dissect, tumor-by-tumor, molecular lesions that are characteristic of distinct cancers. Because of the initial chemical-precipitation step, which purifies and enriches the aberrant protein population as part of PU bead–bound Hsp90 complexes, the method does not require expensive SILAC labeling or two-dimensional gel separation of samples. Instead, protein cargo from PU bead pull-downs is simply eluted in SDS buffer and submitted to standard SDS-PAGE, and then the separated proteins are extracted and trypsinized for LC-MS/MS analysis.
Although this method presents a unique approach to identifying the oncoproteins that maintain the malignant phenotype of tumor cells, one needs to be aware that, similar to other chemical or antibody-based proteomics techniques, it has potential limitations46
. For example, ‘sticky’ or abundant proteins may bind in a nondiscriminatory way to proteins isolated by PU beads. Second, although we have presented several lines of evidence that PU-H71 is specific for Hsp90, one must consider that, at the high concentration of PU-H71 present on the beads, some nonspecific binding to a few non-Hps90 proteins is unavoidable.
Despite these potential limitations, we have used this method to carry out the first global evaluation of Hsp90-dependent aberrant signaling pathways in CML. The Hsp90 interactome identified by PU-H71 affinity purification overlaps substantially with the well-characterized CML signalosome, indicating that this method can identify a large part of the complex web of pathways and proteins that define the molecular basis of this leukemia. When applied to less well-characterized tumor types, this method may provide unpredicted targets for combinatorial therapy.
We believe the functional proteomics method described here will assist identification of the critical proteome subset that is dysregulated in individual tumors, including primary patient specimens. Thus, tumor-specific Hsp90 client profiling could ultimately yield an approach for personalized therapeutic targeting of tumors (Supplementary Fig. 8
Our work also proposes that Hsp90 forms biochemically distinct complexes in cancer cells (Supplementary Fig. 3a
). In this view, a major fraction of cancer cell Hsp90 retains ‘housekeeping’ chaperone functions similar to normal cells, whereas a functionally distinct Hsp90 pool that is enriched or expanded in cancer cells specifically interacts with the oncogenic proteins required to maintain tumor cell survival. Perhaps this Hsp90 fraction represents a cell stress–specific form of chaperone complex that is expanded and constitutively maintained in the tumor cell context. Our data suggest that it may execute functions necessary to maintain the malignant phenotype. One such role is to regulate the folding of mutated (that is, mB-Raf) or chimeric (that is, Bcr-Abl) proteins8,9
. We now present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling complexes.
What distinguishes the PU-H71-binding fraction of Hsp90 from the non–PU-H71-binding fraction? This is a complex question that remains under active investigation. Although both Hsp90α and Hsp90β isoforms are recognized by PU-H71, our data provide evidence for at least one difference between Bcr-Abl–Hsp90 (PU-H71 preferring) and Abl–Hsp90 (PU-H71 nonpreferring) chaperone complexes. That is, Bcr-Abl–Hsp90 chaperone complexes contain a number of cochaperones (suggesting that an active chaperoning process is underway, further supported by the sensitivity of Bcr-Abl to the silencing of Hsp70), whereas Abl–Hsp90 complexes lack associated cochaperones (probably representing sequestered but not actively chaperoned Abl, supported by the insensitivity of Abl to Hsp70 knockdown). Finally, we have observed a differential impact of Hsp90 phosphorylation on PU-H71 and geldanamycin binding. These findings, which are being pursued further, suggest that various Hsp90 inhibitors may be uniquely affected by specific post-translational modifications to the chaperone. Taken together, these preliminary observations suggest that PU-H71 recognizes an Hsp90 fraction that is participating in an active chaperone cycle, and that this characteristic is not necessarily shared by other Hsp90 inhibitors.
Our work uses chemical tools to provide new insights into the heterogeneity of tumor-associated Hsp90 and harnesses the biochemical features of a particular Hsp90 inhibitor to identify tumor-specific biological pathways and proteins. We believe the functional proteomics method described here will allow identification of the critical proteome subset that becomes dysregulated in distinct tumors. This will allow for the identification of new cancer mechanisms, as exemplified by the STAT5 mechanism, the identification of new oncoproteins, as exemplified by CARM1, and the identification of therapeutic targets for the development of rationally combined targeted therapies complementary to Hsp90.