Mutant p53 (mutp53) cancers are surprisingly dependent on their hyperstable mutp53 protein for survival, identifying mutp53 as a potentially significant clinical target. However, exploration of ...effective small molecule therapies targeting mutp53 has barely begun. Mutp53 hyperstabilization, a hallmark of p53 mutation, is cancer cell-specific and due to massive upregulation of the HSP90 chaperone machinery during malignant transformation. We recently showed that stable complex formation between HSP90 and its mutp53 client inhibits E3 ligases MDM2 and CHIP, causing mutp53 stabilization. Histone deacetylase (HDAC) inhibitors (HDACi) are a new class of promising anti-cancer drugs, hyperacetylating histone and non-histone targets. Currently, suberoylanilide hydroxamic acid (SAHA) is the only FDA-approved HDACi. We show that SAHA exhibits preferential cytotoxicity for mutant, rather than wild-type and null p53 human cancer cells. Loss/gain-of-function experiments revealed that although able to exert multiple cellular effects, SAHA's cytotoxicity is caused to a significant degree by its ability to strongly destabilize mutp53 at the level of protein degradation. The underlying mechanism is SAHA's inhibition of HDAC6, an essential positive regulator of HSP90. This releases mutp53 and enables its MDM2- and CHIP-mediated degradation. SAHA also strongly chemosensitizes mutp53 cancer cells for chemotherapy due to its ability to degrade mutp53. This identifies a novel action of SAHA with the prospect of SAHA becoming a centerpiece in mutp53-specific anticancer strategies.
Missense mutations in p53 generate aberrant proteins with abrogated tumour suppressor functions that can also acquire oncogenic gain-of-function activities that promote malignant progression, ...invasion, metastasis and chemoresistance. Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumours, which is the key requisite for the acquisition of gain-of-functions activities. Although currently 11 million patients worldwide live with tumours 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 inactivatable R248Q hotspot mutation (floxQ) to show that tumours depend on sustained mutp53 expression. Upon tamoxifen-induced mutp53 ablation, allotransplanted and autochthonous tumours curb their growth, thus extending animal survival by 37%, and advanced tumours undergo apoptosis and tumour regression or stagnation. The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared with normal tissues, is a major determinant of mutp53 stabilization. We show that long-term HSP90 inhibition significantly extends the survival of mutp53 Q/- (R248Q allele) and H/H (R172H allele) mice by 59% and 48%, respectively, but not their corresponding 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, tumour apoptosis and prevention of T-cell lymphomagenesis. These proof-of-principle data identify mutp53 as an actionable cancer-specific drug target.
Mutant p53 proteins not only lose their tumor-suppressor function but some acquire oncogenic gain of function (GOF). The published mutp53 knock-in (KI) alleles (R172H, R270H, R248W) manifest GOF by ...broader tumor spectrum and more metastasis compared with the p53-null allele, but do not shorten survival. However, whether GOF also occurs with other mutations and whether they are all biologically equal is unknown. To answer this, we created novel humanized mutp53 KI mice harboring the hot spot alleles R248Q and G245S. Intriguingly, their impact was very different. Compared with p53-null mice, R248Q/- mice had accelerated onset of all tumor types and shorter survival, thus unprecedented strong GOF. In contrast, G245S/- mice were similar to null mice in tumor latency and survival. This was associated with a twofold higher T-lymphoma proliferation in R248Q/- mice compared with G245S/- and null mice. Moreover, R248Q/- hematopoietic and mesenchymal stem cells were expanded relative to G245S/- and null mice, the first indication that GOF also acts by perturbing pretumorous progenitor pools. Importantly, these models closely mirror Li-Fraumeni patients who show higher tumor numbers, accelerated onset and shorter tumor-free survival by 10.5 years when harboring codon R248Q mutations as compared with Li-Fraumeni patients with codon G245S mutations or p53 deletions/loss. Conversely, both KI alleles caused a modest broadening of tumor spectrum with enhanced Akt signaling compared with null mice. These models are the first in vivo proof for differential oncogenic strength among p53 GOF alleles, with genotype-phenotype correlations borne out in humans.
The activity of p53 as an inducible transcription factor depends on its rapid nuclear stabilization after stress. However, surprisingly, mechanism(s) that regulate nuclear p53 accumulation are not ...well understood. The current model of stress-induced nuclear accumulation holds that a decrease in p53 nuclear export leads to its nuclear stabilization. We show here that regulated nuclear import of p53 also has a critical function. p53 import is mediated by binding to the importin-
α
3 adapter and is negatively regulated by ubiquitination. p53 harbors several nuclear localization signals (NLS), with the major NLS I located at amino-acids 305–322. We find that direct binding of p53 to importin-
α
3 depends on the positive charge contributed by lysine residues 319–321 within NLS I. The same lysines are also targets of MDM2-mediated ubiquitination. p53 ubiquitination occurs primarily in unstressed cells, but decreases dramatically after stress. Importin-
α
3 preferentially interacts with non-ubiquitinated p53. Thus, under normal growth conditions, ubiquitination of Lys 319–321 negatively regulates p53-importin-
α
3 binding, thereby restraining p53 import. Conversely, stress-induced accumulation of non-ubiquitinated p53 in the cytoplasm promotes interaction with importin-
α
3 and rapid import. In later phases of the stress response, blocked nuclear export also takes effect. We propose that p53 nuclear import defines an important novel level of regulation in the p53-mediated stress response.
The complex apoptotic functions of the p53 tumor suppressor are central to its antineoplastic activity in vivo. Conversely, p53 function is altered or attenuated in one way or another in the majority ...of human cancers. Besides its well-understood action as a transcriptional regulator of multiple apoptotic genes, p53 also exerts a direct pro-apoptotic role at the mitochondria by engaging in protein-protein interactions with anti- and pro-apoptotic Bcl2 family members, thereby executing the shortest known circuitry of p53 death signaling. Nur77, also known as TR3 or NGFI-B, is a unique transcription factor belonging to the orphan nuclear receptor superfamily. Even more extreme than p53, Nur77 can exert opposing biological activities of survival and death. Its activities are regulated by subcellular distribution, expression levels, protein modification and heterodimerization with retinoid X receptor. In cancer cells, Nur77 functions in the nucleus as an oncogenic survival factor, but becomes a potent killer when certain death stimuli induce its migration to mitochondria, where it binds to Bcl2 and conformationally converts it to a killer that triggers cytochrome c release and apoptosis. This review focuses on their unexpected transcription-independent pro-death programs at mitochondria and highlights the remarkable mechanistic similarities between them. Moreover, an accumulating body of evidence provides ample rationale to further investigate how these mitochondrial p53 and Nur77 pathways could become exploitable targets for new cancer therapeutics.
The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by ...cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.
In response to intense stress, the tumor protein p53 (p53) tumor suppressor rapidly mounts a direct mitochondrial death program that precedes transcription-mediated apoptosis. By eliminating severely ...damaged cells, this pathway contributes to tumor suppression as well as to cancer cell killing induced by both genotoxic drugs and non-genotoxic p53-reactivating molecules. Here we have explored the role had in this pathway by the prolyl-isomerase Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1), a crucial transducer of p53's phosphorylation into conformational changes unleashing its pro-apoptotic activity. We show that Pin1 promotes stress-induced localization of p53 to mitochondria both in vitro and in vivo. In particular, we demonstrate that upon stress-induced phosphorylation of p53 on Ser46 by homeodomain interacting protein kinase 2, Pin1 stimulates its mitochondrial trafficking signal, that is, monoubiquitination. This pathway is induced also by the p53-activating molecule RITA, and we demonstrate the strong requirement of Pin1 for the induction of mitochondrial apoptosis by this compound. These findings have significant implications for treatment of p53-expressing tumors and for prospective use of p53-activating compounds in clinics.
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.
In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor ...cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/- glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/- glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/- mice again exhibit normal epithelial architecture by conventional histology. However, using Rosa(LSL-LacZ);WAP-Cre transgenics (LSL-LacZ, lox-stop-lox β-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/- glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/- ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.
The p53 tumor suppressor network plays a fundamental surveillance role in both homeostatic and adaptive cell biology. p53 is one of the most important barriers against malignant derailment of normal ...cells, orchestrating growth arrest, senescence, or cell death by linking many different pathways in response to genotoxic and non-genotoxic insults. p53 is the key broadband sensor for numerous cellular stresses such as DNA damage, hypoxia, oxidative stress, oncogenic signaling, and nucleolar stress. The crucial tumor suppressive and tissue homeostasis activity of p53 is its ability to activate cell death via multiple different pathways. A well-characterized biochemical function of p53 in the regulation of apoptosis is its role as a potent transcriptional regulator. p53 activates a panel of proapoptotic genes from the mitochondrial apoptotic and death receptor programs while repressing antiapoptotic Bcl2 family genes. In addition, over the last 10 y a growing body of evidence has also defined direct extranuclear non-transcriptional p53 activities within mitochondria-mediated cell death pathways that are based on p53 protein accumulation in cytosolic and mitochondrial compartments and protein-protein interactions. To date, transcription-independent p53-mediated cell death regulation has been described for apoptosis, necrosis, and autophagy. Because mitochondrial dysregulation is central to the development of a number of pathologic processes such as cancer and neurodegenerative and age-related diseases, understanding the direct roles of p53 protein in mitochondria has high translational impact and could facilitate the development of novel drug targets to combat these diseases. In this review we will mainly focus on mechanisms of p53-mediated transcription-independent cell death pathways at mitochondria.
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