Nitric Oxide-Releasing Drug Glyceryl Trinitrate Targets JAK2/STAT3 Signaling, Migration and Invasion of Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is a highly aggressive disease with invasive and metastasizing properties associated with a poor prognosis. The STAT3 signaling pathway has shown a pivotal role in cancer cell migration, invasion, metastasis and drug resistance of TNBC cells. IL-6 is a main upstream activator of the JAK2/STAT3 pathway. In the present study we examined the impact of the NO-donor glyceryl trinitrate (GTN) on the activation of the JAK2/STAT3 signaling pathway and subsequent migration, invasion and metastasis ability of TNBC cells through in vitro and in vivo experiments. We used a subtoxic dose of carboplatin and/or recombinant IL-6 to activate the JAK2/STAT3 signaling pathway and its functional outcomes. We found an inhibitory effect of GTN on the activation of the JAK2/STAT3 signaling, migration and invasion of TNBC cells. We discovered that GTN inhibits the activation of JAK2, the upstream activator of STAT3, and mediates the S-nitrosylation of JAK2. Finally, the effect of GTN (Nitronal) on lung metastasis was investigated to assess its antitumor activity in vivo.

Precision medicine in breast cancer: reality or utopia?

Many cancers, including breast cancer, have demonstrated prognosis and support advantages thanks to the discovery of targeted therapies. The advent of these new approaches marked the rise of precision medicine, which leads to improve the diagnosis, prognosis and treatment of cancer. Precision medicine takes into account the molecular and biological specificities of the patient and their tumors that will influence the treatment determined by physicians. This new era of medicine is accessible through molecular genetics platforms, the development of high-speed sequencers and means of analysis of these data. Despite the spectacular results in the treatment of cancers including breast cancer, described in this review, not all patients however can benefit from this new strategy. This seems to be related to the many genetic mutations, which may be different from one patient to another or within the same patient. It comes to give new impetus to the research-both from a technological and biological point of view-to make the hope of precision medicine accessible to all.

Nitroglycerin: a comprehensive review in cancer therapy.

Nitroglycerin (NTG) is a prodrug that has long been used in clinical practice for the treatment of angina pectoris. The biotransformation of NTG and subsequent release of nitric oxide (NO) is responsible for its vasodilatating property. Because of the remarkable ambivalence of NO in cancer disease, either protumorigenic or antitumorigenic (partly dependent on low or high concentrations), harnessing the therapeutic potential of NTG has gain interest to improve standard therapies in oncology. Cancer therapeutic resistance remains the greatest challenge to overcome in order to improve the management of cancer patients. As a NO releasing agent, NTG has been the subject of several preclinical and clinical studies used in combinatorial anticancer therapy. Here, we provide an overview of the use of NTG in cancer therapy in order to foresee new potential therapeutic avenues.

Cellular inhibitor of apoptosis protein-1 (cIAP1) can regulate E2F1 transcription factor-mediated control of cyclin transcription.

The inhibitor of apoptosis protein cIAP1 (cellular inhibitor of apoptosis protein-1) is a potent regulator of the tumor necrosis factor (TNF) receptor family and NF-κB signaling pathways in the cytoplasm. However, in some primary cells and tumor cell lines, cIAP1 is expressed in the nucleus, and its nuclear function remains poorly understood. Here, we show that the N-terminal part of cIAP1 directly interacts with the DNA binding domain of the E2F1 transcription factor. cIAP1 dramatically increases the transcriptional activity of E2F1 on synthetic and CCNE promoters. This function is not conserved for cIAP2 and XIAP, which are cytoplasmic proteins. Chromatin immunoprecipitation experiments demonstrate that cIAP1 is recruited on E2F binding sites of the CCNE and CCNA promoters in a cell cycle- and differentiation-dependent manner. cIAP1 silencing inhibits E2F1 DNA binding and E2F1-mediated transcriptional activation of the CCNE gene. In cells that express a nuclear cIAP1 such as HeLa, THP1 cells and primary human mammary epithelial cells, down-regulation of cIAP1 inhibits cyclin E and A expression and cell proliferation. We conclude that one of the functions of cIAP1 when localized in the nucleus is to regulate E2F1 transcriptional activity.

Protein Kinase Inhibitor-Mediated Immunoprophylactic and Immunotherapeutic Control of Colon Cancer.

Immunotherapy has allowed major advances in oncology in the past years, in particular with the development of immune checkpoint inhibitors, but the clinical benefits are still limited, particularly in colorectal cancer (CRC). Our scientific approach is based on the search for innovative immunotherapy with a final goal that aims to induce an effective antitumor immune response in CRC. Here, we focused on a multikinase inhibitor, H89. We carried out in vivo experiments based on syngeneic mouse models of colon cancer in BALB/c mice and chemically colon tumorigenesis. Flow cytometry, RNAseq, RT-qPCR, antibody-specific immune cell depletion, and Western blot were used to identify the immune cell type involved in the preventive and antitumor activity of H89. We demonstrated that H89 delays colon oncogenesis and prevents tumor growth. This latter effect seems to involve NK cells. H89 also inhibits colon tumor growth in a T-cell-dependent manner. Analysis of the immune landscape in the tumor microenvironment showed an increase of CD4+ Th1 cells and CD8+ cytotoxic T cells but a decrease of CD4+ Treg cell infiltration. Mechanistically, we showed that H89 could promote naïve CD4+ T-cell differentiation into Th1, a decrease in Treg differentiation, and an increase in CD8+ T-cell activation and cytotoxicity ex vivo. Furthermore, H89 induced overexpression of genes involved in antitumor immune response, such as IL-15RA, which depletion counteracts the antitumor effect of H89. We also found that H89 regulated Akt/PP2A pathway axis, involved in TCR and IL-15 signaling transduction. Our findings identify the H89 as a potential strategy for immune system activation leading to the prevention and treatment of CRC.

Protein kinase inhibitor-based cancer therapies: Considering the potential of nitric oxide (NO) to improve cancer treatment.

The deregulation of a wide variety of protein kinases is associated with cancer cell initiation and tumor progression. Owing to their indispensable function in signaling pathways driving malignant cell features, protein kinases constitute major therapeutic targets in cancer. Over the past two decades, intense efforts in drug development have been dedicated to this field. The development of protein kinase inhibitors (PKIs) have been a real breakthrough in targeted cancer therapy. Despite obvious successes across patients with different types of cancer, the development of PKI resistance still prevails. Combination therapies are part of a comprehensive approach to address the problem of drug resistance. The therapeutic use of nitric oxide (NO) donors to bypass PKI resistance in cancer has never been tested in clinic yet but several arguments suggest that the combination of PKIs and NO donors may exert a potential anticancer effect. The present review summarized the current state of knowledge on common targets to both PKIs and NO. Herein, we attempt to provide the rationale underlying a potential combination of PKIs and NO donors for future directions and design of new combination therapies in cancer.

Senescence and Cancer: Role of Nitric Oxide (NO) in SASP.

Cellular senescence is a cell state involved in both physiological and pathological processes such as age-related diseases and cancer. While the mechanism of senescence is now well known, its role in tumorigenesis still remains very controversial. The positive and negative effects of senescence on tumorigenesis depend largely on the diversity of the senescent phenotypes and, more precisely, on the senescence-associated secretory phenotype (SASP). In this review, we discuss the modulatory effect of nitric oxide (NO) in SASP and the possible benefits of the use of NO donors or iNOS inducers in combination with senotherapy in cancer treatment.

Glyceryl trinitrate­induced cytotoxicity of docetaxel­resistant prostatic cancer cells is associated with differential regulation of clusterin.

Metastatic castration resistant prostate cancer (mCRPC) relapse due to acquired resistance to chemotherapy, such as docetaxel, remains a major threat to patient survival. Resistance of mCRPC to docetaxel can be associated with elevated levels of soluble clusterin (sCLU) and growth differentiation factor­15 (GDF­15). Any strategies aiming to modulate sCLU and/or GDF­15 in docetaxel­resistant prostate cancer cells present a therapeutic interest. The present study reports the cytotoxic effect of a nitric oxide donor, glyceryl trinitrate (GTN), on docetaxel­resistant mCRPC human cell lines and demonstrates that GTN displays greater inhibition of cell viability toward docetaxel­resistant mCRPC cells than on mCRPC cells. It is also demonstrated that GTN modulates the level of expression of clusterin (CLU) which is dependent of GDF­15, two markers associated with docetaxel resistance in prostate cancer. The results indicate that GTN represses the level of expression of the cytoprotective isoform of CLU (sCLU) and can increase the level of expression of the cytotoxic isoform (nuclear CLU) in docetaxel resistant cells. Furthermore, it was observed that GTN differentially regulates the level of the precursor form of GDF­15 between resistant and parental cells, and that recombinant GDF­15 can modulate the expression of CLU isoforms and counteract GTN­induced cytotoxicity in resistant cells. A link was established between GDF­15 and the expression of CLU isoforms. The present study thus revealed GTN as a potential therapeutic strategy to overcome docetaxel­resistant mCRPC.

S-Nitrosylation of cIAP1 Switches Cancer Cell Fate from TNFα/TNFR1-Mediated Cell Survival to Cell Death.

TNFα is a prominent proinflammatory cytokine and a critical mediator for the development of many types of cancer such as breast, colon, prostate, cervical, skin, liver, and chronic lymphocytic leukemia. Binding of TNFα to TNFR1 can lead to divergent signaling pathways promoting predominantly NF-κB activation but also cell death. We report here that the nitric oxide (NO) donor glyceryl trinitrate (GTN) converts TNFα, generated from immune cells or cancer cells stimulated by chemotherapy, into a prodeath mediator in colon and mammary cancer cells. GTN-mediated S-nitrosylation of cIAP1 on cysteines 571 and 574 inhibited its E3 ubiquitin ligase activity, which in turn reduced Lys63-linked ubiquitination of RIP1 and initiated assembly of a death complex. These findings provide insights into how NO can harness advantageous aspects of inflammation in cancer and provide new therapeutic strategies.Significance: Combination of an NO donor with chemotherapeutic drug-induced TNFα represents a potentially valuable anticancer strategy. Cancer Res; 78(8); 1948-57. ©2018 AACR.

The role of XAF1 in cancer.

The inhibitors of apoptosis (IAP) proteins have emerged as important cancer targets. The cellular control of IAP expression is regulated by survival signaling pathways and by a variety of known intrinsic antagonists. Among these antagonists, the X-linked IAP-associated factor (XAF)1 is unique in its control of IAP function and in its ability to sensitize cancer cells to apoptosis. Studies have demonstrated that XAF1 is strongly pro-apoptotic, is inducible by IFN and is a tumor suppressor. Thus, this antagonist may have significant value in the treatment of cancer.

Silencing of the XAF1 gene by promoter hypermethylation in cancer cells and reactivation to TRAIL-sensitization by IFN-beta.

BACKGROUND: XIAP-associated factor 1 (XAF1) is a putative tumor suppressor that exerts its proapoptotic effects through both caspase-dependent and -independent means. Loss of XAF1 expression through promoter methylation has been implicated in the process of tumorigenesis in a variety of cancers. In this report, we investigated the role of basal xaf1 promoter methylation in xaf1 expression and assessed the responsiveness of cancer cell lines to XAF1 induction by IFN-beta. METHODS: We used the conventional bisulfite DNA modification and sequencing method to determine the methylation status in the CpG sites of xaf1 promoter in glioblastoma (SF539, SF295), neuroblastoma (SK-N-AS) and cervical carcinoma (HeLa) cells. We analysed the status and incidence of basal xaf1 promoter methylation in xaf1 expression in non-treated cells as well as under a short or long exposure to IFN-beta. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-beta-mediated sensitization to TRAIL-induced cell death. RESULTS: We found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-beta across the four cancer cell lines studied. At the basal level, aberrant promoter methylation was linked to xaf1 gene silencing. After a short exposure, the IFN-beta-mediated reactivation of xaf1 gene expression was related to the degree of basal promoter methylation. However, in spite of continued promoter hypermethylation, we find that IFN-beta induced a transient xaf1 expression, that in turn, was followed by promoter demethylation upon a prolonged exposure. Importantly, we demonstrated for the first time that IFN-beta-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-beta-mediated TRAIL sensitivity. CONCLUSION: Together, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-beta treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer.

HSP27 is a ubiquitin-binding protein involved in I-kappaBalpha proteasomal degradation.

HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-alpha). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-kappaB by degrading its main inhibitor, I-kappaBalpha. HSP27 overexpression increases NF-kappaB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and interleukin 1beta. HSP27 does not affect I-kappaBalpha phosphorylation but enhances the degradation of phosphorylated I-kappaBalpha by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-kappaBalpha. A protein complex that includes HSP27, phosphorylated I-kappaBalpha, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-kappaBalpha. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-kappaB activity.

Exploration of Fas S-Nitrosylation by the Biotin Switch Assay.

S-nitrosylation is the covalent attachment of nitric oxide radical to the thiol side chain of cysteine. The death receptor Fas/CD95 can be S-nitrosylated in cancer cell lines by NO donors or iNOS activation. This posttranslational modification (PTM) induces Fas aggregation into lipid rafts and enhances FasL-mediated signaling and apoptosis. In this report, we describe the detection of Fas S-nitrosylation by the most commonly used method, the biotin switch assay (BSA) technique, that allows the detection of this very labile covalent modification in cells or tissues. Briefly, this technique relies on the ability of ascorbate to reduce the covalent bond between the NO radical and the protein, allowing the exchange of the NO radical with a thiol reactive biotin-HPDP. The biotinylated proteins are then easily purified by using NeutrAvidin resin, separated by SDS-PAGE resolution and analyzed by Western blotting.

[IAPs and cancer: The NO strikes back]. / IAP et cancer : le NO contre-attaque.

TITLE: IAP et cancer : le NO contre-attaque. ABSTRACT: Dans le cadre du Master 2 de l'université EPHE-PSL (Master Sciences du vivant, cursus IMaGHE, parcours Physiopathologie intégrative [PPI]), des étudiants se sont confrontés à la rédaction d'une nouvelle scientifique. Ces étudiants ayant choisi une spécialisation en cancérologie, l'équipe pédagogique leur a proposé de faire une synthèse d'articles sur deux thématiques : 1) les protéines IAP et un mécanisme original de régulation de leur activité par S-nitrosylation et 2) la séparase, dont un article paru récemment dans Nature montre qu'elle jouerait un rôle inattendu dans la protection des cellules contre la transformation tumorale. Organisés en binôme ou trinôme, les étudiants ont rédigé deux nouvelles qui soulignent l'intérêt des travaux analysés, ainsi que leur originalité. Ils se sont pleinement investis dans cette tâche et ont su faire preuve d'un bel esprit de synthèse. Ils ont apprécié cet exercice nouveau pour eux, mais qui leur a permis d'avoir un aperçu de la publication scientifique inhérente au métier de chercheur auquel ils se destinent.

[NO and cancer: itinerary of a double agent]. / Itinéraire d'un agent double - NO, S-nitrosylation et cancer.

Protein S-nitrosylation is now recognized as a ubiquitous regulatory mechanism. Like any post-translational modifications, S-nitrosylation is critical for the control of numerous cellular processes. It is now clear that S-nitrosylation is playing a double game, enhancing or inhibiting the tumor growth or the induction of cell death. Thanks to research aimed at demonstrating NO cytotoxic effects, new therapeutic strategies based on NO donor drugs have emerged. Although therapeutic NO donors can target a large number of proteins, the cellular mechanism is still not fully understood. This review reflects the current state of knowledge on S-nitrosylated proteins that take part of the oncogenic and apoptotic signaling, putting forward proteins with potential interest in cancer therapy.

IAPs: Mediators of Oncogenesis and Targets for Anticancer Therapy.

The inhibitor of apoptosis (IAP) family members are potent regulators of cell homeostasis able to regulate several fundamental cellular processes that include cell death, cell proliferation, cell differentiation, and inflammation. Regarding this broad spectrum of activity, it is now becoming clear that some members of the family possess oncogenic properties. Analysis of genomic database from tumor sequencing studies has revealed a number of genetic alterations affecting some IAP genes and resulting in gain or loss of function. In this review, we discuss the importance of IAP alterations in cell transformation and their link with key oncogenic pathways, focusing on nuclear factor-kappa B (NF-κB)-activating signaling pathways. Then we highlight the therapeutic potential of IAP antagonists and nitric oxide (NO) donors as inhibitors of NF-κB in anticancer therapy.

Role Of S-Nitrosylation In The Extrinsic Apoptotic Signalling Pathway In Cancer.

One of the key features of tumour cells is the acquisition of resistance to apoptosis. Thus, determining therapeutic strategies that circumvent apoptotic resistance and result in tumor regression is a challenge. One strategy to induce apoptosis is to activate death receptor signalling pathways. Members of the Tumor Necrosis Factor TNF-family death receptors ligand (TRAIL, FasL and TNF-α) can originate from immune and non-immune cells. Death receptors, engaged by cognate ligands, can initiate multiple signaling pathways, which can generate diverse outcomes, including non-apoptosis-related signal. Knowledge on the molecular mechanisms (that determine death or survival of tumour cells) following exposure to the TNF-family death receptors ligands have demonstrated that post-translational modifications of the signaling pathway components play a critical role in determining cell fate. Cell death can be sensed by nitric oxide (NO) in a wide variety of tumour cells. S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO moiety, has emerged as an important post-translational regulation for the TNF-family death receptor signaling pathways. It has been demonstrated that death receptor DR4 (TRAIL-R1) becomes S-nitrosylated and promotes apoptosis following a specific NO donor treatment (Tang et al., 2006). Then, our group has shown that S-nitrosylation of Fas, following glyceryl trinitrate (GTN) exposure, promotes redistribution of the receptor to lipid rafts, formation of the death-inducing signal complex (DISC), and induction of cell death. Finally, I will discuss our recent efforts to decipher regulatory mechanism of the TNF-α/TNFR1 signalling cell death pathway by S-nitrosylation following GTN treatment.

S-Nitrosylation in TNF superfamily signaling pathway: Implication in cancer.

One of the key features of tumor cells is the acquisition of resistance to apoptosis. Thus, novel therapeutic strategies that circumvent apoptotic resistance and result in tumor elimination are needed. One strategy to induce apoptosis is to activate death receptor signaling pathways. In the tumor microenvironment, stimulation of Fas, Death receptor 4 (DR4) and tumor necrosis factor receptor 1 (TNFR1) can initiate multiple signaling pathways driving either tumor promotion or elimination. Nitric oxide (NO) is an important signaling molecule now understood to play a dual role in cancer biology. More and more attention is directed toward the role displayed by S-nitrosylation, the incorporation of an NO moiety to a cysteine thiol group, in promoting cell death in tumor cells. Protein post-translation modification by S-nitrosylation has decisive roles in regulating signal-transduction pathways. In this review, we summarize several examples of protein modification by S-nitrosylation that regulate signaling pathways engaged by members of the TNF superfamily (Fas ligand (FasL), Tumor-necrosis-factor-related apoptosis inducing ligand (TRAIL) and TNFalpha (TNFα)) and the way it influences cell fate decisions.

Bcl-2 proteins: targets and tools for chemosensitisation of tumor cells.

Proteins of the Bcl-2 family share one or several Bcl-2 homology (BH) regions and behave as pro- or anti-apoptotic proteins. Prosurvival members such as Bcl-2 and Bcl-X(L) are supposed to preserve mitochondrial outer membrane integrity, thus preventing the release of soluble apoptogenic molecules. Pro-apoptotic members include BH3-only proteins that act as sensors of cellular damage and initiate the death process and Bax-like proteins that act downstream of BH3-only proteins to permeabilise the mitochondrial outer membrane. Whether BH3-only proteins directly activate Bax-like proteins or prevent prosurvival members of the family from inhibiting Bax-like proteins or both remains a matter of controversy. Expression of these proteins is altered in various human tumours and this abnormal expression may contribute to oncogenesis and tumour cell resistance to anticancer drug-induced cell death. Based on these observations, prosurvival proteins are attractive intracellular targets for inducing tumour cell death or sensitising tumour cells to death induced by chemotherapeutic drugs. The use of 18-mer antisense oligonucleotides (G3139 or Genasense) targeting the first six codons of bcl-2 mRNA is currently developed in clinics with phase I studies demonstrating that thrombocytopenia may be the main dose-limiting side effect. This strategy, that efficiently decreases Bcl-2 protein expression in some tumour cells, is currently tested in phase II and phase III trials. Alternative approaches to achieve the functional knock-out of Bcl-2 include the use of either peptides mimicking the BH3 domain of Bcl-2-related proteins or more stable, non peptidic BH3 mimetics and the pharmacological modulation of the post-translational modifications of the protein.

Analyzing markers of apoptosis in vitro.

Cell death by apoptosis was first identified based on morphological changes reproduced with great fidelity in cells of widely different origin when exposed to a death stimulus. These changes include condensation of the cytosol and the nuclear chromatin, blebbing of the plasma membrane, and cell fragmentation into corpses that are engulfed by neighboring cells. Apoptotic cells demonstrate various levels of DNA fragmentation and exposed phosphatidylserine on the outer leaflet of their plasma membrane. Most apoptotic pathways converge on the mitochondria, inducing the disruption of the mitochondrial trans-membrane potential and the release of soluble molecules from mitochondrial inter-membrane space. One of these molecules is cytochrome c, which, in the cytosol, activates proteases of the caspase family. This chapter suggests methods to identify these characteristic morphological and biochemical events, and cell-free systems that can be used to identify the molecular pathways leading to the death phenotype.