Giovanni Sorrentino

p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner

Significance Accumulating evidence has underscored the role of cytosolic p53 in promoting cell death. Different reports have revealed that p53 participates in apoptosis induction by acting directly at mitochondria. However, because p53 can mediate apoptosis without its DNA-binding domain (the domain proposed to be fundamental for the targeting of p53 to mitochondria), the mitochondrial localization of p53 is likely not the only transcription-independent mechanism by which p53 promotes apoptosis. Here we demonstrate that p53 at the endoplasmic reticulum (ER) and at mitochondria-associated membranes, interacting with sarco/ER Ca2+-ATPase pumps, modulates ER–mitochondria cross-talk and, in turn, Ca2+-dependent apoptosis. The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca2+ homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca2+-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca2+ load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca2+ overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca2+ signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca2+ signal-dependent apoptosis.

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.

YAP enhances the pro-proliferative transcriptional activity of mutant p53 proteins

Mutant p53 proteins are present in more than half of human cancers. Yes‐associated protein (YAP) is a key transcriptional regulator controlling organ growth, tissue homeostasis, and cancer. Here, we report that these two determinants of human malignancy share common transcriptional signatures. YAP physically interacts with mutant p53 proteins in breast cancer cells and potentiates their pro‐proliferative transcriptional activity. We found YAP as well as mutant p53 and the transcription factor NF‐Y onto the regulatory regions of cyclin A, cyclin B, and CDK1 genes. Either mutant p53 or YAP depletion down‐regulates cyclin A, cyclin B, and CDK1 gene expression and markedly slows the growth of diverse breast cancer cell lines. Pharmacologically induced cytoplasmic re‐localization of YAP reduces the expression levels of cyclin A, cyclin B, and CDK1 genes both in vitro and in vivo. Interestingly, primary breast cancers carrying p53 mutations and displaying high YAP activity exhibit higher expression levels of cyclin A, cyclin B, and CDK1 genes when compared to wt‐p53 tumors.

Regulation of mitochondrial apoptosis by Pin1 in cancer and neurodegeneration

Mitochondria are sensitive and efficient organelles that regulate essential biological processes including: energy metabolism, decoding and transduction of intracellular signals, and balance between cell death and survival. Of note, dysfunctions in mitochondrial physiology are a general hallmark of cancer cells, leading to transformation-related features such as altered cellular metabolism, survival under stress conditions and reduced apoptotic response to chemotherapy. Mitochondrial apoptosis is a finely regulated process that derives from activation of multiple signaling networks. A crucial biochemical requirement for transducing pro-apoptotic stimuli is represented by kinase-dependent phosphorylation cascades. In this context a pivotal role is played by the prolyl-isomerase Pin1, which translates Ser/Thr-Pro phosphorylation into conformational changes able to modify the activities of its substrates. In this review we will discuss the impact of Pin1 in regulating various aspects of apoptosis in different biological contexts with particular emphasis on cancer and neurodegenerative diseases.

MDP, a database linking drug response data to genomic information, identifies dasatinib and statins as a combinatorial strategy to inhibit YAP/TAZ in cancer cells

Targeted anticancer therapies represent the most effective pharmacological strategies in terms of clinical responses. In this context, genetic alteration of several oncogenes represents an optimal predictor of response to targeted therapy. Integration of large-scale molecular and pharmacological data from cancer cell lines promises to be effective in the discovery of new genetic markers of drug sensitivity and of clinically relevant anticancer compounds. To define novel pharmacogenomic dependencies in cancer, we created the Mutations and Drugs Portal (MDP, http://mdp.unimore.it), a web accessible database that combines the cell-based NCI60 screening of more than 50,000 compounds with genomic data extracted from the Cancer Cell Line Encyclopedia and the NCI60 DTP projects. MDP can be queried for drugs active in cancer cell lines carrying mutations in specific cancer genes or for genetic markers associated to sensitivity or resistance to a given compound. As proof of performance, we interrogated MDP to identify both known and novel pharmacogenomics associations and unveiled an unpredicted combination of two FDA-approved compounds, namely statins and Dasatinib, as an effective strategy to potently inhibit YAP/TAZ in cancer cells.

Glucocorticoid receptor signalling activates YAP in breast cancer

The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance.

Mechanical cues control mutant p53 stability through a mevalonate–RhoA axis

Tumour-associated p53 missense mutants act as driver oncogenes affecting cancer progression, metastatic potential and drug resistance (gain-of-function)1. Mutant p53 protein stabilization is a prerequisite for gain-of-function manifestation; however, it does not represent an intrinsic property of p53 mutants, but rather requires secondary events2. Moreover, mutant p53 protein levels are often heterogeneous even within the same tumour, raising questions on the mechanisms that control local mutant p53 accumulation in some tumour cells but not in their neighbours2,3. By investigating the cellular pathways that induce protection of mutant p53 from ubiquitin-mediated proteolysis, we found that HDAC6/Hsp90-dependent mutant p53 accumulation is sustained by RhoA geranylgeranylation downstream of the mevalonate pathway, as well as by RhoA- and actin-dependent transduction of mechanical inputs, such as the stiffness of the extracellular environment. Our results provide evidence for an unpredicted layer of mutant p53 regulation that relies on metabolic and mechanical cues.Ingallina et al. show that mutant p53 is protected from degradation in response to matrix stiffness in a manner dependent on RhoA geranylgeranylation and actomyosin dynamics.

Translocation of signalling proteins to the plasma membrane revealed by a new bioluminescent procedure.

BackgroundActivation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors.High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.ResultsHere we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca2+)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca2+ concentration [Ca2+] difference between bulk cytosolic and the sub-plasma membrane rim.ConclusionThis approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane.Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation.Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.

GDA, a web-based tool for Genomics and Drugs integrated analysis

Abstract Several major screenings of genetic profiling and drug testing in cancer cell lines proved that the integration of genomic portraits and compound activities is effective in discovering new genetic markers of drug sensitivity and clinically relevant anticancer compounds. Despite most genetic and drug response data are publicly available, the availability of user-friendly tools for their integrative analysis remains limited, thus hampering an effective exploitation of this information. Here, we present GDA, a web-based tool for Genomics and Drugs integrated Analysis that combines drug response data for >50 800 compounds with mutations and gene expression profiles across 73 cancer cell lines. Genomic and pharmacological data are integrated through a modular architecture that allows users to identify compounds active towards cancer cell lines bearing a specific genomic background and, conversely, the mutational or transcriptional status of cells responding or not-responding to a specific compound. Results are presented through intuitive graphical representations and supplemented with information obtained from public repositories. As both personalized targeted therapies and drug-repurposing are gaining increasing attention, GDA represents a resource to formulate hypotheses on the interplay between genomic traits and drug response in cancer. GDA is freely available at http://gda.unimore.it/.

The stiff RhoAd from mevalonate to mutant p53

Mutant p53 oncoproteins (mutp53), produced as result of missense mutations in the TP53 gene, actively promote tumour aggressive traits, metastatic dissemination and chemoresistance. Massive accumulation of mutp53 protein occurs in tumour cells [1] and is required for its oncogenic gain-of-function, indicating pharmacological destabilization of mutp53 as a potential anticancer therapeutic strategy [2]. In a recent issue of Nature Cell Biology [3], we reported that mechanical inputs, transduced by RhoA-dependent cytoskeletal tension, sustain mutp53 protein stability and that blocking this axis with mevalonate (MVA) pathway inhibitors curbs mutp53 accumulation in tumours (Fig. 1). Relying on an extended protein interactome, which includes transcriptional regulators not bound by the wildtype counterpart, mutp53 drives tumour cell metabolic rewiring, migration/invasion, acquisition of stem traits and chemoresistance. In this context, mutp53 becomes constitutively stable, due to its engagement in complexes with the Hsp90 chaperone machinery, which prevents mutp53 poly-ubiquitination and proteasomal degradation [4]. Pharmacological destabilization of mutp53 by blocking Hsp90 with new generation inhibitors has proven effective to cause tumour regression in vivo [2]. Supporting the clinical efficacy of this strategy, HSP90 inhibitors were found to synergise with CCPT (concurrent cisplatin radiotherapy) in HNSCC cancers with mutant TP53 status [5]. This evidence further incites the quest for efficient and well-tolerated drugs targeting mutp53 stability as future chemotherapeutic treatments. Drug repositioning approaches represent a valid strategy to obtain hints on mechanisms sustaining oncogene activation, and to identify molecules able to interfere with these processes. Work by our group [3] and by others [6] highlighted that statins, a class of MVA pathway inhibitors and a very common drug used in the clinic for treatment of cardiovascular diseases, elicit mutp53 destabilization and reducing cancer cell proliferation. The MVA pathway is a conserved metabolic route that uses acetyl-CoA to produce cholesterol and other key biomolecules, some of which are required to support tumour development and progression. Specifically, the isoprenoid geranylgeranyl-pyrophosphate (GGPP) produced along the MVA pathway, is essential for post-translational modification and membrane anchoring of many proteins involved in aggressive cancer phenotypes. Among them, the small GTPase RhoA links ECM rigidity to intracellular actomyosin tension, acting as a mechanotransducer to drive tumour cell survival, proliferation and progression. In our work, we demonstrated that GGPP acts to stabilise the interaction of mutp53 with Hsp90, thus preventing its degradation (Fig. 1). We showed that this effect requires the histone deacetylase HDAC6, a direct activator of Hsp90 [7]. Interestingly, HDAC6 is controlled by changes in cytoskeleton dynamics [8], critical events in the crosstalk of transformed cells with the tumour microenvironment. Tumours display altered mechanotransduction compared to normal tissues, as cancer-associated fibrosis generates a dense and mechanically rigid extracellular matrix (ECM) leading to integrin clustering and activation in focal adhesions. These complexes induce RhoAdependent actin remodelling and actomyosin contractility (Fig. 1). It has become increasingly clear that mechanical cues presented to cells as a result of tissue stiffening, favour cancer development and progression. Interestingly, the levels of mutp53 appear frequently heterogenous within tumour tissues, with mutp53 over-expressing foci associated with fibrotic regions [9], suggesting that mutp53 stability may be locally influenced by tissue rigidity. * Giannino Del Sal delsal@lncib.it