SUMMARYMissense mutations in p53 generate aberrant proteins with abrogated tumor suppressor functions that can also acquire oncogenic gain-of-functions (GOF) that promote malignant progression, invasion, metastasis and chemoresistance1–5. Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumors, which is the key requisite for GOF6–8. Although currently 11 million patients worldwide live with tumors expressing highly stabilized mutp53, it is unknown whether mutp53 is a therapeutic target in vivo.Here we use a novel mutp53 mouse model expressing an inactivatible R248Q hotspot mutation (floxQ) to show that tumors depend on sustained mutp53 expression. Upon Tamoxifen-induced mutp53 ablation, allo-transplanted and autochthonous tumors curb their growth, thus extending animal survival by 37%, and advanced tumors undergo apoptosis and tumor regression or stagnation.The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared to normal tissues, is a major determinant of mutp53 stabilization9–12. We show that long-term HSP90 inhibition significantly extends the survival of mutp53 Q/−2 and H/H (R172H allele3) mice by 59% and 48%, respectively, but not their respective p53−/− littermates. This mutp53-dependent drug effect occurs in H/H mice treated with 17DMAG+SAHA and in H/H and Q/− mice treated with the potent Hsp90 inhibitor ganetespib. Notably, drug activity correlates with induction of mutp53 degradation, tumor apoptosis and prevention of T-lymphomagenesis. These proof-of-principle data identify mutp53 as an actionable cancer-specific drug target.

The tight control of wild-type (wt) p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53 (mutp53), which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wtp53 tumors. Importantly, mutp53 hyperstabilization is critical for mutp53′s oncogenic gain-of function in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutp53 knockin mice established that normal mutp53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutp53. We find that in human cancer cells endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wtp53, the many mutp53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wtp53 cancer cells, we show that in mutp53 cancer cells this HSP90 interaction blocks the endogenous MDM2 and CHIP E3 ligase activity. Interference with HSP90 either by RNAi against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacological inhibition of Hsp90 activity with 17AAG destroys the complex, liberates mutp53 and reactivates endogenous MDM2 and CHIP to degrade mutp53. Of note, 17AAG induces a stronger viability loss in mutp53 than in wtp53 cancer cells. Our data supports the rationale that suppression of mutp53 levels in vivo in established cancers might achieve clinically significant effects.

In several human cancer cell lines, HSP90 inhibitors destabilize macrophage inhibitory factor protein; systemic treatment with an HSP90 inhibitor slows tumor growth and extends overall survival in a mouse model of HER2-positive human breast cancer.

Overexpression of the human epidermal growth factor receptor-2 (HER2) in breast cancer strongly correlates with aggressive tumors and poor prognosis. Recently, a positive correlation between HER2 and MIF (macrophage migration inhibitory factor, a tumor-promoting protein and heat-shock protein 90 (HSP90) client) protein levels was shown in cancer cells. However, the underlying mechanistic link remained unknown. Here we show that overexpressed HER2 constitutively activates heat-shock factor 1 (HSF1), the master transcriptional regulator of the inducible proteotoxic stress response of heat-shock chaperones, including HSP90, and a crucial factor in initiation and maintenance of the malignant state. Inhibiting HER2 pharmacologically by Lapatinib (a dual HER2/epidermal growth factor receptor inhibitor) or CP724.714 (a specific HER2 inhibitor), or by knockdown via siRNA leads to inhibition of phosphoactivated Ser326 HSF1, and subsequently blocks the activity of the HSP90 chaperone machinery in HER2-overexpressing breast cancer lines. Consequently, HSP90 clients, including MIF, AKT, mutant p53 and HSF1 itself, become destabilized, which in turn inhibits tumor proliferation. Mechanistically, HER2 signals via the phosphoinositide-3-kinase (PI3K)–AKT– mammalian target of rapamycin (mTOR) axis to induce activated pSer326 HSF1. Heat-shock stress experiments confirm this functional link between HER2 and HSF1, as HER2 (and PI3K) inhibition attenuate the HSF1-mediated heat-shock response. Importantly, we confirmed this axis in vivo. In the mouse model of HER2-driven breast cancer, ErbB2 inhibition by Lapatinib strongly suppresses tumor progression, and this is associated with inactivation of the HSF1 pathway. Moreover, ErbB2-overexpressing cancer cells derived from a primary mouse ErbB2 tumor also show HSF1 inactivation and HSP90 client destabilization in response to ErbB2 inhibition. Furthermore, in HER2-positive human breast cancers HER2 levels strongly correlate with pSer326 HSF1 activity. Our results show for the first time that HER2/ErbB2 overexpression controls HSF1 activity, with subsequent stabilization of numerous tumor-promoting HSP90 clients such as MIF, AKT and HSF1 itself, thereby causing a robust promotion in tumor growth in HER2-positive breast cancer.