Hsp90 Inhibition on the Western Blot: N‐Terminal Versus C‐Terminal Inhibitors in Breast Cancer

Heat shock protein 90 (Hsp90) is an established and clinically validated anticancer target. By regulating the folding and maturation of diverse client proteins, it is indirectly involved in carcinogenesis. Key breast cancer oncogenic drivers, including estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are established Hsp90 clients, making breast cancer models widely used for Hsp90 inhibitor evaluation. However, N-terminal inhibitors are well known to induce a heat shock response (HSR), representing a major limitation in cancer therapy. This has driven the development of alternative modalities, such as C-terminal inhibitors, which in several cases avoid HSR induction. To evaluate Hsp90 inhibitors and characterize their proteomic effects, most studies employ western blotting (WB) as the primary method. On WB, different inhibitors are tested across various concentrations, time points, and breast cancer cell lines for their effects on client, heat shock, and other proteins. This generates extensive data sets that outline the expected WB performance of Hsp90 inhibitors. In this review, we focus on 10 protein families that are commonly used to assess Hsp90 inhibition in in vitro breast cancer models and represent all 10 cancer hallmarks. We provide basic structural and functional information on these families and comprehensively describe the effects of N- and C-terminal inhibitors on their levels. We summarize WB-based observations for more than 100 Hsp90 inhibitors, outlining the characteristic effects of N- and C-terminal inhibition, highlighting opportunities for future research, and ending with a suggestion for the WB method design based on best practices observed in literature.