Venturina Stagni

Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression

We identified Caspase‐8 as a new substrate for Src kinase. Phosphorylation occurs on Tyr380, situated in the linker region between the large and the small subunits of human Procaspase‐8, and results in downregulation of Caspase‐8 proapoptotic function. Src activation triggers Caspase‐8 phosphorylation on Tyr380 and impairs Fas‐induced apoptosis. Accordingly, Src failed to protect Caspase‐8‐defective human cells in which a Caspase‐8‐Y380F mutant is expressed from Fas‐induced cell death. Remarkably, Src activation upon EGF‐receptor stimulation triggers endogenous Caspase‐8 phosphorylation and prevents Fas‐induced apoptosis. Tyr380 is phosphorylated also in human colon cancers where Src is aberrantly activated. These data provide the first evidence for a direct role of tyrosine phosphorylation in the control of caspases and reveal a new mechanism through which tyrosine kinases inhibit apoptosis and participate in tumor progression.

Identification of a Critical Tyrosine Residue in Caspase 8 That Promotes Cell Migration

Caspase 8 is a critical upstream initiator of programmed cell death but, paradoxically, has also been shown to promote cell migration. Here, we show that tyrosine 380 in the linker loop of human caspase 8 is a critical switch determining caspase 8 function. Our studies show that, in addition to its cytosolic distribution, caspase 8 is recruited to lamella of migrating cells. Although the catalytic domain of caspase 8 is sufficient for recruitment and promotion of cell migration, catalytic activity per se is not required. Instead, we find that integrin-mediated adhesion promotes caspase 8 phosphorylation on tyrosine 380. Accordingly, mutation of this site compromises localization to the periphery and the potentiation of cell migration. Mechanistically, this linker region of caspase 8 acts as a Src homology 2 binding site. In particular, tyrosine 380 is critical for interaction with Src homology 2 domains. The results identify a novel mechanism by which caspase 8 is recruited to the lamella of a migrating cell, promoting cell migration independent of its protease activity.

ATM-depletion in breast cancer cells confers sensitivity to PARP inhibition.

BackgroundMutations in the DNA damage response (DDR) factors, breast cancer 1 (BRCA1) and BRCA2, sensitize tumor cells to poly(ADP-ribose) polymerase (PARP) inhibitors. The ataxia telangiectasia mutated (ATM) kinase is a key DDR protein whose heterozygous germline mutation is a moderate–risk factor for developing breast cancer. In this study, we examined whether ATM inactivation in breast cancer cell lines confers sensitivity to PARP inhibitors.MethodsWild-type BRCA1/2 breast cancer cells (i.e., MCF-7 and ZR-75-1 lines) were genetically manipulated to downregulate ATM expression then assayed for cytostaticity/cytotoxicity upon treatment with PARP inhibitors, olaparib and iniparib.ResultsWhen ATM-depleted cells and their relative controls were treated with olaparib (a competitive PARP-1/2 inhibitor) and iniparib (a molecule originally described as a covalent PARP-1 inhibitor) a different response to the two compounds was observed. ATM-depletion sensitized both MCF-7 and ZR-75-1 cells to olaparib-treatment, as assessed by short and long survival assays and cell cycle profiles. In contrast, iniparib induced only a mild, ATM-dependent cytostatic effect in MCF-7 cells whereas ZR-75-1 cells were sensitive to this drug, independently of ATM inactivation. These latest results might be explained by recent observations indicating that iniparib acts with mechanisms other than PARP inhibition.ConclusionsThese data indicate that ATM-depletion can sensitize breast cancer cells to PARP inhibition, suggesting a potential in the treatment of breast cancers low in ATM protein expression/activity, such as those arising in mutant ATM heterozygous carriers.

ATM kinase activity modulates cFLIP protein levels: potential interplay between DNA damage signalling and TRAIL-induced apoptosis

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a potent tool to trigger apoptosis in cancer therapy. However, since ∼60% of tumour cell lines and most primary cancers are resistant to TRAIL-induced apoptosis, several combined therapy approaches aimed to sensitize cells to TRAIL have been developed. One of the major targets of these approaches are cFLIP proteins as they interfere with the initiation of apoptosis induction by TRAIL, are over-expressed in many cancers and their down-regulation enhances TRAIL sensitivity. Although, DNA-damaging agents such as 5-fluorouracil (5-FU), etoposide and adriamycin have been successfully employed due to their ability to trigger cFLIP(L) and cFLIP(s) down-regulation the molecular mechanisms underneath their action have been only partially elucidated. We have recently identified ataxia telangiectasia mutated (ATM) as a modulator of cFLIP(L) and cFLIP(S) protein levels in the DNA damage response. Here, we provide genetic evidence that ATM kinase activity is required to trigger 5-FU- and neocarzinostatin-dependent cFLIP(L) and cFLIP(S) down-regulation, which in turn sensitize hepatocellular carcinoma (HCC) cell lines to TRAIL. ATM activity triggers cFLIP proteins down-regulation in HCC cells independently on p53 and enhances cFLIP(L) ubiquitination in response to DNA damage. Therefore, we propose that ATM kinase mediates the interplay between DNA damage and death receptor signalling and suggest that expression of catalytically competent ATM in tumour cells may play a key role for successful combinatorial use of TRAIL receptor agonists and DNA-damaging drugs in cancer therapy.

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolisms regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics.

Oxygen sensing is impaired in ATM-defective cells.

The transcription factor hypoxia-inducible factor 1α (HIF-1α) is a master regulator of cell adaptation to decreasing oxygen levels. High oxygen tension promotes proteosomal degradation of HIF-1α via a pathway that requires hydroxylation of prolines 402 and 564. Low oxygen tension, hypoxia, inactivates the hydroxylases responsible for these modifications through a mechanism that is not fully understood but appears to require mitochondrial respiration and production of reactive oxygen species, ROS. Cells from individuals affected by ataxia telangiectasia syndrome have an impaired mitochondrial activity and a constitutive oxidative stress. Here we show that, in these cells, HIF-1α is efficiently degraded even in condition of low oxygen tension. Mechanistically this depends from a blunted increase in intracellular concentration of ROS in response to hypoxia which in turn is due to an increased cellular capacity of buffering ROS. We suggest that regulation of HIF-1α stability may depend on fold change of ROS relative to the basal level more than on their absolute value. Since elevated oxidative stress is a hallmark of many human disorders our finding may be relevant to different pathologies.

ATM kinase sustains HER2 tumorigenicity in breast cancer

ATM kinase preserves genomic stability by acting as a tumour suppressor. However, its identification as a component of several signalling networks suggests a dualism for ATM in cancer. Here we report that ATM expression and activity promotes HER2-dependent tumorigenicity in vitro and in vivo. We reveal a correlation between ATM activation and the reduced time to recurrence in patients diagnosed with invasive HER2-positive breast cancer. Furthermore, we identify ATM as a novel modulator of HER2 protein stability that acts by promoting a complex of HER2 with the chaperone HSP90, therefore preventing HER2 ubiquitination and degradation. As a consequence, ATM sustains AKT activation downstream of HER2 and may modulate the response to therapeutic approaches, suggesting that the status of ATM activity may be informative for the treatment and prognosis of HER2-positive tumours. Our findings provide evidence for ATMs tumorigenic potential revising the canonical role of ATM as a pure tumour suppressor.

Tug of War between Survival and Death: Exploring ATM Function in Cancer

Ataxia-telangiectasia mutated (ATM) kinase is a one of the main guardian of genome stability and plays a central role in the DNA damage response (DDR). The deregulation of these pathways is strongly linked to cancer initiation and progression as well as to the development of therapeutic approaches. These observations, along with reports that identify ATM loss of function as an event that may promote tumor initiation and progression, point to ATM as a bona fide tumor suppressor. The identification of ATM as a positive modulator of several signalling networks that sustain tumorigenesis, including oxidative stress, hypoxia, receptor tyrosine kinase and AKT serine-threonine kinase activation, raise the question of whether ATM function in cancer may be more complex. This review aims to give a complete overview on the work of several labs that links ATM to the control of the balance between cell survival, proliferation and death in cancer.

ATM kinase activity modulates ITCH E3-ubiquitin ligase activity.

Ataxia Telangiectasia Mutated (ATM) kinase, a central regulator of the DNA damage response, regulates the activity of several E3-ubiquitin ligases, and the ubiquitination-proteasome system is a consistent target of ATM. ITCH is an E3-ubiquitin ligase that modulates the ubiquitination of several targets, therefore participating to the regulation of several cellular responses, such as the DNA damage response, tumor necrosis factorα (TNFα), Notch and Hedgehog signaling, and the differentiation of ‘naive’ lymphocytes into T helper type 2 cells. Here we uncover ATM as a novel positive modulator of ITCH E3-ubiquitin ligase activity. A single residue on ITCH protein, S161, which is part of an ATM SQ consensus motif, is required for ATM-dependent activation of ITCH. ATM activity enhances ITCH enzymatic activity, which in turn drives the ubiquitination and degradation of c-FLIP-L and c-Jun, previously identified as ITCH substrates. Importantly, ATM-deficient mice show resistance to hepatocyte cell death, similarly to Itch-deficient animals, providing in vivo genetic evidence for this circuit. Our data identify ITCH as a novel component of the ATM-dependent signaling pathway and suggest that the impairment of the correct functionality of ITCH caused by Atm deficiency may contribute to the complex clinical features linked to Ataxia Telangiectasia.

Met acts through Abl to regulate p53 transcriptional outcomes and cell survival in the developing liver.

BACKGROUND & AIMS Genetic studies indicate that distinct signaling modulators are each necessary but not individually sufficient for embryonic hepatocyte survival in vivo. Nevertheless, how signaling players are interconnected into functional circuits and how they coordinate the balance of cell survival and death in developing livers are still major unresolved issues. In the present study, we examined the modulation of the p53 pathway by HGF/Met in embryonic livers. METHODS We combined pharmacological and genetic approaches to biochemically and functionally evaluate p53 pathway modulation in primary embryonic hepatocytes and in developing livers. RT-PCR arrays were applied to investigate the selectivity of p53 transcriptional response triggered by Met. RESULTS Met recruits p53 to regulate the liver developmental program, by qualitatively modulating its transcriptional properties: turning on the Mdm2 survival gene, while keeping death and cell-cycle arrest genes Pmaip1 and p21 silent. We investigated the mechanism leading to p53 regulation by Met and found that Abl and p38MAPK are required for p53 phosphorylation on S(389), Mdm2 upregulation, and hepatocyte survival. Alteration of this signaling mechanism switches p53 properties, leading to p53-dependent cell death in embryonic livers. RT-PCR array studies affirmed the ability of the Met-Abl-p53 axis to modulate the expression of distinct genes that can be regulated by p53. CONCLUSIONS A signaling circuit involving Abl and p38MAPK is required downstream of Met for the survival of embryonic hepatocytes, via qualitative regulation of the p53 transcriptional response, by switching its proapoptotic into survival properties.