Targeting basal-like breast cancer via purine-scaffold Hsp90 inhibitors

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Basal-like breast cancers (BLC) are highly aggressive tumors with a relatively poor prognosis that account for approximately 15% of sporadic human breast cancer (BC). Sporadic BLC do not express the estrogen receptor (ER) and do not overproduce HER2 protein. Thus, therapeutics targeting ER or HER2 currently used in treating BC are unlikely to be useful for these patients, leaving them with no efficacious treatment options. In malignant cells, including those of the breast, the transformed phenotype is maintained by heat shock protein 90 (Hsp90). This chaperone interacts in a transformation-specific manner with aberrant proteins involved in and required for driving malignancy. Association of Hsp90 with these oncoproteins allows them to function in the deregulated state and appears to be essential for their transforming activity. Therefore, we hypothesize that Hsp90 inhibitors, by interacting specifically with the chaperone function, may potently promote the destabilization and degradation of aberrant proteins that maintain transformation in BLC cells. For this study, several Hsp90 inhibitors such as PU-H71, a purine-scaffold inhibitor currently in advanced preclinical development, the ansamycin 17AAG and the purine-scaffold CNF-2024, both in clinical evaluation in patients with advanced cancers, were chosen. Here we present the effects of these inhibitors in MDA-MB-468, HCC1806 and MDA-MB-231 BLC cells. We find that these cells are highly sensitive to the purine-scaffold inhibitors at concentrations in high agreement with their affinity for Hsp90, but remain partially resistant to 17AAG treatment. 17AAG binds efficiently to Hsp90 isolated from these cells, suggesting other factors such as multidrug efflux mechanisms or intracellular drug metabolism as culprits for its lessened cellular activity. The Hsp90 inhibitors potently inhibited the proliferation of these cells and downregulated AKT and Raf-1, two nodal oncoproteins regulating the survival and proliferative advantage of BLC cells. A dramatic accumulation of cells containing sub-G1 DNA content, and influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis, was seen with increasing doses of drugs. Notably, we observed a dramatic increase in caspase-3,7 activity upon addition of this agent to BLC cells, effect associated with cleavage of caspase-3 and PARP. We also found that treatment with PU-H71 significantly increased the percentage of MDA-MB-468 cells in mitosis. When administered in vivo to mice bearing MDA-MB-468 human breast cancer xenografted tumors, PU-H71 and CNF-2024 resulted in pharmacologically relevant concentrations and accordingly, in modulation of Hsp90-client proteins in tumors and consequent tumor growth inhibition. These data suggest the purine-scaffold Hsp90 inhibitors as potential therapeutics in the treatment patients with BLC.