Improving survival by exploiting tumour dependence on stabilized mutant p53 for treatment.

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.

UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib.

HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC50) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC50>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.

HER2/ErbB2 activates HSF1 and thereby controls HSP90 clients including MIF in HER2-overexpressing 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.

p63 is a prosurvival factor in the adult mammary gland during post-lactational involution, affecting PI-MECs and ErbB2 tumorigenesis.

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 ß-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.

Mitochondrial death functions of p53.

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.

ΔNp63 regulates select routes of reprogramming via multiple mechanisms.

Somatic cells can be converted into induced pluripotent stem cells (iPSCs) by forced expression of various combinations of transcription factors, but the molecular mechanisms of reprogramming are poorly understood. Specifically, evidence that the reprogramming process can take many distinct routes only begins to emerge. It is definitively established that p53 deficiency greatly enhances reprogramming, revealing p53's barrier function for induced pluripotency, but the role of its homologs p63 and p73 are unknown. Here we report that in stark contrast to p53, p73 has no role in reprogramming. However, p63 is an enabling (rather than a barrier) factor for Oct4, Sox2 and Klf4 (OSK) and Oct4 and Sox2 (OS), but not for Oct4 and Klf4 (OK) reprogramming of mouse embryonic fibroblasts. Specifically, p63 is essential during reprogramming for maximum efficiency, albeit not for the ability to reprogram per se, and is dispensable for maintaining stability and pluripotency of established iPSC colonies. ΔNp63, but not TAp63, is the principal isoform involved. Loss of p63 can affect reprogramming via several mechanisms such as reduced expression of mesenchymal-epithelial transition and pluripotency genes, hypoproliferation and loss of the most reprogrammable cell populations. During OSK and OS reprogramming, different mechanisms seem to be critical, such as regulation of epithelial and pluripotency genes in OSK reprogramming versus regulation of proliferation in OS reprogramming. Finally, our data reveal three different routes of reprogramming by OSK, OS or OK, based on their differential p63 requirements for iPSC efficiency and pluripotency marker expression. This supports the concept that many distinct routes of reprogramming exist.

Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis.

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 prolyl-isomerase Pin1 activates the mitochondrial death program of p53.

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.

Two-factor reprogramming of somatic cells to pluripotent stem cells reveals partial functional redundancy of Sox2 and Klf4.

Ectopic expression of defined sets of transcription factors in somatic cells enables them to adopt the qualities of pluripotency. Mouse embryonic fibroblasts (MEFs) are the classic target cell used to elucidate the core principles of nuclear reprogramming. However, their phenotypic and functional heterogeneity represents a major hurdle for mechanistic studies aimed at defining the molecular nature of cellular plasticity. We show that reducing the complexity of MEFs by flow cytometry allows the isolation of discrete cell subpopulations that can be efficiently reprogrammed to pluripotency with fewer genes. Using these FACS-sorted cells, we performed a systematic side-by-side analysis of the reprogramming efficiency with different two- and three-factor combinations of Oct4, Sox2 and Klf4. We show that introduction of exogenous Oct4 with either Sox2 or Klf4 does not directly convert MEFs to a pluripotent state. Instead, each combination of factors disrupts the normal cellular homeostasis and establishes transient states characterized by the concurrent expression of mixed lineage markers. These cells convert into induced pluripotent stem cells in a stochastic fashion. Our data suggest that there is a partial functional redundancy between Sox2 and Klf4 in the disruption of cellular homeostasis and activation of regulatory networks that define pluripotency.

SAHA shows preferential cytotoxicity in mutant p53 cancer cells by destabilizing mutant p53 through inhibition of the HDAC6-Hsp90 chaperone axis.

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.

Blockade of Hsp90 by 17AAG antagonizes MDMX and synergizes with Nutlin to induce p53-mediated apoptosis in solid tumors.

Strategies to induce p53 activation in wtp53-retaining tumors carry high potential in cancer therapy. Nutlin, a potent highly selective MDM2 inhibitor, induces non-genotoxic p53 activation. Although Nutlin shows promise in promoting cell death in hematopoietic malignancies, a major roadblock is that most solid cancers do not undergo apoptosis but merely reversible growth arrest. p53 inhibition by unopposed MDMX is one major cause for apoptosis resistance to Nutlin. The Hsp90 chaperone is ubiquitously activated in cancer cells and supports oncogenic survival pathways, many of which antagonize p53. The Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) is known to induce p53-dependent apoptosis. We show here that in multiple difficult-to-kill solid tumor cells 17AAG modulates several critical components that synergize with Nutlin-activated p53 signaling to convert Nutlin's transient cytostatic response into a cytotoxic killing response in vitro and in xenografts. Combined with Nutlin, 17AAG destabilizes MDMX, reduces MDM2, induces PUMA and inhibits oncogenic survival pathways, such as PI3K/AKT, which counteract p53 signaling at multiple levels. Mechanistically, 17AAG interferes with the repressive MDMX-p53 axis by inducing robust MDMX degradation, thereby markedly increasing p53 transcription compared with Nutlin alone. To our knowledge Nutlin+17AAG represents the first effective pharmacologic knockdown of MDMX. Our study identifies 17AAG as a promising synthetic lethal partner for a more efficient Nutlin-based therapy.

p73 is an essential regulator of neural stem cell maintenance in embryonal and adult CNS neurogenesis.

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.

Stress-mediated nuclear stabilization of p53 is regulated by ubiquitination and importin-alpha3 binding.

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-alpha3 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-alpha3 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-alpha3 preferentially interacts with non-ubiquitinated p53. Thus, under normal growth conditions, ubiquitination of Lys 319-321 negatively regulates p53-importin-alpha3 binding, thereby restraining p53 import. Conversely, stress-induced accumulation of non-ubiquitinated p53 in the cytoplasm promotes interaction with importin-alpha3 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.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

The alpha/beta carboxy-terminal domains of p63 are required for skin and limb development. New insights from the Brdm2 mouse which is not a complete p63 knockout but expresses p63 gamma-like proteins.

p63, an ancestral transcription factor of the p53 family, has three C-terminal isoforms whose relative in vivo functions are elusive. The p63 gene is essential for skin and limb development, as vividly shown by two independent global knockout mouse models. Both strains, although constructed differently, have identical and severe phenotypes, characterized by absent epidermis and hindlimbs and only rudimentary forelimbs at birth. Here we show that mice from one model, Brdm2, express normal levels of truncated p63 proteins that contain the DNA binding and oligomerization domain but lack the long carboxy-terminal SAM (sterile alpha-motif) and post-SAM domains that are specific for the alpha and beta isoforms. As such, transcriptionally active p63 proteins from Brdm2 mice resemble the naturally occurring p63gamma isoforms, which of all the p63 isoforms most closely resemble p53. Thus, Brdm2 mice are p63alpha/beta isoform-specific knockout mice, gaining unexpected new importance. Our studies identify that p63alpha/beta but not p63gamma are absolutely required for proper skin and limb development.

Rb function is required for E1A-induced S-phase checkpoint activation.

It is widely accepted that adenoviral E1A exerts its influence on recipient cells through binding to the retinoblastoma (Rb) family proteins, followed by a global release of E2F factors from pocket-protein control. Our study challenges this simple paradigm by demonstrating previously unappreciated complexity. We show that E1A-expressing primary and transformed cells are characterized by the persistence of Rb-E2F1 complexes. We provide evidence that E1A causes Rb stabilization by interfering with its proteasomal degradation. Functional experiments supported by biochemical data reveal not only a dramatic increase in Rb and E2F1 protein levels in E1A-expressing cells but also demonstrate their activation throughout the cell cycle. We further show that E1A activates an Rb- and E2F1-dependent S-phase checkpoint that attenuates the growth of cells that became hyperploid through errors in mitosis and supports the fidelity DNA replication even in the absence of E2F complexes with other Rb family proteins, thereby functionally substituting for the loss of p53. Our results support the essential role of Rb and E2F1 in the regulation of genomic stability and DNA damage checkpoints.