The unfolded protein response-glutathione metabolism axis: A novel target of a cycloruthenated complexes bypassing tumor resistance mechanisms.

Platinum-based drugs remain the reference treatment for gastric cancer (GC). However, the frequency of resistance, due to mutations in TP53 or alterations in the energy and redox metabolisms, impairs the efficacy of current treatments, highlighting the need for alternative therapeutic options. Here, we show that a cycloruthenated compound targeting the redox metabolism, RDC11, induces higher cytotoxicity than oxaliplatin in GC cells and is more potent in reducing tumor growth in vivo. Detailed investigations into the mode of action of RDC11 indicated that it targets the glutathione (GSH) metabolism, which is an important drug resistance mechanism. We demonstrate that cycloruthenated complexes regulate the expression of enzymes of the transsulfuration pathway via the Unfolded Protein Response (UPR) and its effector ATF4. Furthermore, RDC11 induces the expression of SLC7A11 encoding for the cystine/glutamate antiporter xCT. These effects lead to a lower cellular GSH content and elevated oxygen reactive species production, causing the activation of a caspase-independent apoptosis. Altogether, this study provides the first evidence that cycloruthenated complexes target the GSH metabolism, neutralizing thereby a major resistance mechanism towards platinum-based chemotherapies and anticancer immune response.

Altering relative metal-binding affinities in multifunctional Metallochaperones for mutant p53 reactivation.

The p53 protein plays a major role in cancer prevention, and over 50% of cancer diagnoses can be attributed to p53 malfunction. p53 incorporates a structural Zn site that is required for proper protein folding and function, and in many cases point mutations can result in loss of the Zn2+ ion, destabilization of the tertiary structure, and eventual amyloid aggregation. Herein, we report a series of compounds designed to act as small molecule stabilizers of mutant p53, and feature Zn-binding fragments to chaperone Zn2+ to the metal depleted site and restore wild-type (WT) function. Many Zn metallochaperones (ZMCs) have been shown to generate intracellular reactive oxygen species (ROS), likely by chelating redox-active metals such as Fe2+/3+ and Cu+/2+ and undergoing associated Fenton chemistry. High levels of ROS can result in off-target effects and general toxicity, and thus, careful tuning of ligand Zn2+ affinity, in comparison to the affinity for other endogenous metals, is important for selective mutant p53 targeting. In this work we show that by using carboxylate donors in place of pyridine we can change the relative Zn2+/Cu2+ binding ability in a series of ligands, and we investigate the impact of donor group changes on metallochaperone activity and overall cytotoxicity in two mutant p53 cancer cell lines (NUGC3 and SKGT2).

A novel ΔNp63-dependent immune mechanism improves prognosis of HPV-related head and neck cancer.

Background: Deconvoluting the heterogenous prognosis of Human Papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OSCC) is crucial for enhancing patient care, given its rapidly increasing incidence in western countries and the adverse side effects of OSCC treatments. Methods: Transcriptomic data from HPV-positive OSCC samples were analyzed using unsupervised hierarchical clustering, and clinical relevance was evaluated using Kaplan-Meier analysis. HPV-positive OSCC cell line models were used in functional analyses and phenotypic assays to assess cell migration and invasion, response to cisplatin, and phagocytosis by macrophages in vitro. Results: We found, by transcriptomic analysis of HPV-positive OSCC samples, a ΔNp63 dependent molecular signature that is associated with patient prognosis. ΔNp63 was found to act as a tumor suppressor in HPV-positive OSCC at multiple levels. It inhibits cell migration and invasion, and favors response to chemotherapy. RNA-Seq analysis uncovered an unexpected regulation of genes, such as DKK3, which are involved in immune response-signalling pathways. In agreement with these observations, we found that ΔNp63 expression levels correlate with an enhanced anti-tumor immune environment in OSCC, and ΔNp63 promotes cancer cell phagocytosis by macrophages through a DKK3/NF-κB-dependent pathway. Conclusion: Our findings are the first comprehensive identification of molecular mechanisms involved in the heterogeneous prognosis of HPV-positive OSCC, paving the way for much-needed biomarkers and targeted treatment.

Correction: Structural tuning of organoruthenium compounds allows oxidative switch to control ER stress pathways and bypass multidrug resistance.

[This corrects the article DOI: 10.1039/C6SC00268D.].

Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C.

The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.

Histone Deacetylase Functions in Gastric Cancer: Therapeutic Target?

Gastric cancer (GC) is one of the most aggressive cancers. Therapeutic treatments are based on surgery combined with chemotherapy using a combination of platinum-based agents. However, at metastatic stages of the disease, survival is extremely low due to late diagnosis and resistance mechanisms to chemotherapies. The development of new classifications has not yet identified new prognostic markers for clinical use. The studies of epigenetic processes highlighted the implication of histone acetylation status, regulated by histone acetyltransferases (HATs) and by histone deacetylases (HDACs), in cancer development. In this way, inhibitors of HDACs (HDACis) have been developed and some of them have already been clinically approved to treat T-cell lymphoma and multiple myeloma. In this review, we summarize the regulations and functions of eighteen HDACs in GC, describing their known targets, involved cellular processes, associated clinicopathological features, and impact on survival of patients. Additionally, we resume the in vitro, pre-clinical, and clinical trials of four HDACis approved by Food and Drug Administration (FDA) in cancers in the context of GC.

The EXTREME Regimen Associating Cetuximab and Cisplatin Favors Head and Neck Cancer Cell Death and Immunogenicity with the Induction of an Anti-Cancer Immune Response.

(1) Background: The first line of treatment for recurrent/metastatic Head and Neck Squamous Cell Carcinoma (HNSCC) has recently evolved with the approval of immunotherapies that target the anti-PD-1 immune checkpoint. However, only about 20% of the patients display a long-lasting objective tumor response. The modulation of cancer cell immunogenicity via a treatment-induced immunogenic cell death is proposed to potentially be able to improve the rate of patients who respond to immune checkpoint blocking immunotherapies. (2) Methods: Using human HNSCC cell line models and a mouse oral cancer syngeneic model, we have analyzed the ability of the EXTREME regimen (combination therapy using the anti-EGFR cetuximab antibody and platinum-based chemotherapy) to modify the immunogenicity of HNSCC cells. (3) Results: We showed that the combination of cetuximab and cisplatin reduces cell growth through both cell cycle inhibition and the induction of apoptotic cell death independently of p53. In addition, different components of the EXTREME regimen were found to induce, to a variable extent, and in a cell-dependent manner, the emission of mediators of immunogenic cell death, including calreticulin, HMGB1, and type I Interferon-responsive chemokines. Interestingly, cetuximab alone or combined with the IC50 dose of cisplatin can induce an antitumor immune response in vivo, but not when combined with a high dose of cisplatin. (4) Conclusions: Our observations suggest that the EXTREME protocol or cetuximab alone are capable, under conditions of moderate apoptosis induction, of eliciting the mobilization of the immune system and an anti-tumor immune response in HNSCC.

Ultrasound and Transcriptomics Identify a Differential Impact of Cisplatin and Histone Deacetylation on Tumor Structure and Microenvironment in a Patient-Derived In Vivo Model of Gastric Cancer.

The reasons behind the poor efficacy of transition metal-based chemotherapies (e.g., cisplatin) or targeted therapies (e.g., histone deacetylase inhibitors, HDACi) on gastric cancer (GC) remain elusive and recent studies suggested that the tumor microenvironment could contribute to the resistance. Hence, our objective was to gain information on the impact of cisplatin and the pan-HDACi SAHA (suberanilohydroxamic acid) on the tumor substructure and microenvironment of GC, by establishing patient-derived xenografts of GC and a combination of ultrasound, immunohistochemistry, and transcriptomics to analyze. The tumors responded partially to SAHA and cisplatin. An ultrasound gave more accurate tumor measures than a caliper. Importantly, an ultrasound allowed a noninvasive real-time access to the tumor substructure, showing differences between cisplatin and SAHA. These differences were confirmed by immunohistochemistry and transcriptomic analyses of the tumor microenvironment, identifying specific cell type signatures and transcription factor activation. For instance, cisplatin induced an "epithelial cell like" signature while SAHA favored a "mesenchymal cell like" one. Altogether, an ultrasound allowed a precise follow-up of the tumor progression while enabling a noninvasive real-time access to the tumor substructure. Combined with transcriptomics, our results underline the different intra-tumoral structural changes caused by both drugs that impact differently on the tumor microenvironment.

Bypassing the Resistance Mechanisms of the Tumor Ecosystem by Targeting the Endoplasmic Reticulum Stress Pathway Using Ruthenium- and Osmium-Based Organometallic Compounds: An Exciting Long-Term Collaboration with Dr. Michel Pfeffer.

Metal complexes have been used to treat cancer since the discovery of cisplatin and its interaction with DNA in the 1960's. Facing the resistance mechanisms against platinum salts and their side effects, safer therapeutic approaches have been sought through other metals, including ruthenium. In the early 2000s, Michel Pfeffer and his collaborators started to investigate the biological activity of organo-ruthenium/osmium complexes, demonstrating their ability to interfere with the activity of purified redox enzymes. Then, they discovered that these organo-ruthenium/osmium complexes could act independently of DNA damage and bypass the requirement for the tumor suppressor gene TP53 to induce the endoplasmic reticulum (ER) stress pathway, which is an original cell death pathway. They showed that other types of ruthenium complexes-as well complexes with other metals (osmium, iron, platinum)-can induce this pathway as well. They also demonstrated that ruthenium complexes accumulate in the ER after entering the cell using passive and active mechanisms. These particular physico-chemical properties of the organometallic complexes designed by Dr. Pfeffer contribute to their ability to reduce tumor growth and angiogenesis. Taken together, the pioneering work of Dr. Michel Pfeffer over his career provides us with a legacy that we have yet to fully embrace.

Isoforms of the p53 Family and Gastric Cancer: A Ménage à Trois for an Unfinished Affair.

Gastric cancer is one of the most aggressive cancers, with a median survival of 12 months. This illustrates its complexity and the lack of therapeutic options, such as personalized therapy, because predictive markers do not exist. Thus, gastric cancer remains mostly treated with cytotoxic chemotherapies. In addition, less than 20% of patients respond to immunotherapy. TP53 mutations are particularly frequent in gastric cancer (±50% and up to 70% in metastatic) and are considered an early event in the tumorigenic process. Alterations in the expression of other members of the p53 family, i.e., p63 and p73, have also been described. In this context, the role of the members of the p53 family and their isoforms have been investigated over the years, resulting in conflicting data. For instance, whether mutations of TP53 or the dysregulation of its homologs may represent biomarkers for aggressivity or response to therapy still remains a matter of debate. This uncertainty illustrates the lack of information on the molecular pathways involving the p53 family in gastric cancer. In this review, we summarize and discuss the most relevant molecular and clinical data on the role of the p53 family in gastric cancer and enumerate potential therapeutic innovative strategies.

The atypical cadherin MUCDHL antagonizes colon cancer formation and inhibits oncogenic signaling through multiple mechanisms.

Cadherins form a large and pleiotropic superfamily of membranous proteins sharing Ca2+-binding repeats. While the importance of classic cadherins such as E- or N-cadherin for tumorigenesis is acknowledged, there is much less information about other cadherins that are merely considered as tissue-specific adhesion molecules. Here, we focused on the atypical cadherin MUCDHL that stood out for its unusual features and unique function in the gut. Analyses of transcriptomic data sets (n > 250) established that MUCDHL mRNA levels are down-regulated in colorectal tumors. Importantly, the decrease of MUCDHL expression is more pronounced in the worst-prognosis subset of tumors and is associated with decreased survival. Molecular characterization of the tumors indicated a negative correlation with proliferation-related processes (e.g., nucleic acid metabolism, DNA replication). Functional genomic studies showed that the loss of MUCDHL enhanced tumor incidence and burden in intestinal tumor-prone mice. Extensive structure/function analyses revealed that the mode of action of MUCDHL goes beyond membrane sequestration of ß-catenin and targets through its extracellular domain key oncogenic signaling pathways (e.g., EGFR, AKT). Beyond MUCDHL, this study illustrates how the loss of a gene critical for the morphological and functional features of mature cells contributes to tumorigenesis by dysregulating oncogenic pathways.

HDAC4 Levels Control Sensibility toward Cisplatin in Gastric Cancer via the p53-p73/BIK Pathway.

Gastric cancer (GC) remains a health issue due to the low efficiency of therapies, such as cisplatin. This unsatisfactory situation highlights the necessity of finding factors impacting GC sensibility to therapies. We analyzed the cisplatin pangenomic response in cancer cells and found HDAC4 as a major epigenetic regulator being inhibited. HDAC4 mRNA repression was partly mediated by the cisplatin-induced expression of miR-140. At a functional level, HDAC4 inhibition favored cisplatin cytotoxicity and reduced tumor growth. Inversely, overexpression of HDAC4 inhibits cisplatin cytotoxicity. Importantly, HDAC4 expression was found to be elevated in gastric tumors compared to healthy tissues, and in particular in specific molecular subgroups. Furthermore, mutations in HDAC4 correlate with good prognosis. Pathway analysis of genes whose expression in patients correlated strongly with HDAC4 highlighted DNA damage, p53 stabilization, and apoptosis as processes downregulated by HDAC4. This was further confirmed by silencing of HDAC4, which favored cisplatin-induced apoptosis characterized by cleavage of caspase 3 and induction of proapoptotic genes, such as BIK, in part via a p53-dependent mechanism. Altogether, these results reveal HDAC4 as a resistance factor for cisplatin in GC cells that impacts on patients' survival.

A redox ruthenium compound directly targets PHD2 and inhibits the HIF1 pathway to reduce tumor angiogenesis independently of p53.

Targeting specific tumor metabolic needs represents an actively investigated therapeutic strategy to bypass tumor resistance mechanisms. In this study, we describe an original approach to impact the cancer metabolism by exploiting the redox properties of a ruthenium organometallic compound. This organometallic complex induced p53-independent cytotoxicity and reduced size and vascularization of patients-derived tumor explants that are resistant to platinum drugs. At the molecular level, the ruthenium complex altered redox enzyme activities and the intracellular redox state by increasing the NAD+/NADH ratio and ROS levels. Pathway analysis pointed to HIF-1 as a top deregulated metabolite pathway. Unlike cisplatin, treatment with the ruthenium complex decreased HIF1A protein levels and expression of HIF1A target genes. The rapid downregulation of HIF1A protein levels involved a direct interaction of the ruthenium compound with the redox enzyme PHD2, a HIF1A master regulator. HIF1A inhibition led to decreased angiogenesis in patient-derived xenografted using fragments of primary human colon tumors. Altogether, our results show that a ruthenium compound impacts metabolic pathways acting as anticancer agents in colon cancer via an original mechanism of action that affects redox enzymes differently than platinum-based drugs.

A ruthenium anticancer compound interacts with histones and impacts differently on epigenetic and death pathways compared to cisplatin.

Ruthenium complexes are considered as potential replacements for platinum compounds in oncotherapy. Their clinical development is handicapped by a lack of consensus on their mode of action. In this study, we identify three histones (H3.1, H2A, H2B) as possible targets for an anticancer redox organoruthenium compound (RDC11). Using purified histones, we confirmed an interaction between the ruthenium complex and histones that impacted on histone complex formation. A comparative study of the ruthenium complex versus cisplatin showed differential epigenetic modifications on histone H3 that correlated with differential expression of histone deacetylase (HDAC) genes. We then characterized the impact of these epigenetic modifications on signaling pathways employing a transcriptomic approach. Clustering analyses showed gene expression signatures specific for cisplatin (42%) and for the ruthenium complex (30%). Signaling pathway analyses pointed to specificities distinguishing the ruthenium complex from cisplatin. For instance, cisplatin triggered preferentially p53 and folate biosynthesis while the ruthenium complex induced endoplasmic reticulum stress and trans-sulfuration pathways. To further understand the role of HDACs in these regulations, we used suberanilohydroxamic acid (SAHA) and showed that it synergized with cisplatin cytotoxicity while antagonizing the ruthenium complex activity. This study provides critical information for the characterization of signaling pathways differentiating both compounds, in particular, by the identification of a non-DNA direct target for an organoruthenium complex.

Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase Trim63.

Mechanisms of muscle atrophy are complex and their understanding might help finding therapeutic solutions for pathologies such as amyotrophic lateral sclerosis (ALS). We meta-analyzed transcriptomic experiments of muscles of ALS patients and mouse models, uncovering a p53 deregulation as common denominator. We then characterized the induction of several p53 family members (p53, p63, p73) and a correlation between the levels of p53 family target genes and the severity of muscle atrophy in ALS patients and mice. In particular, we observed increased p63 protein levels in the fibers of atrophic muscles via denervation-dependent and -independent mechanisms. At a functional level, we demonstrated that TAp63 and p53 transactivate the promoter and increased the expression of Trim63 (MuRF1), an effector of muscle atrophy. Altogether, these results suggest a novel function for p63 as a contributor to muscular atrophic processes via the regulation of multiple genes, including the muscle atrophy gene Trim63.

Structural tuning of organoruthenium compounds allows oxidative switch to control ER stress pathways and bypass multidrug resistance.

Multidrug resistance (MDR) is a major impediment to the success of chemotherapy in many cancer types. One particular MDR mechanism is the inherent or acquired adaptation of the cellular survival pathways that render malignant cells resistant to apoptotic cell death. Since most drugs act through apoptosis, compounds capable of inducing alternative forms of programmed cell death (PCD) can potentially be harnessed to bypass MDR. We investigated two organoruthenium complexes, RAS-1H and RAS-1T, and demonstrated that although they both induced non-apoptotic PCD through ER stress pathways, their modes-of-action were drastically different despite modest structural variations. RAS-1T acted through ROS-mediated ER stress while RAS-1H was ROS-independent. We further showed that they were more efficacious against apoptosis-resistant cells compared to clinical drugs including oxaliplatin. This work provides the basis for underpinning ER stress modulation using metal complexes to bypass apoptosis resistance.

Regulation of a Notch3-Hes1 pathway and protective effect by a tocopherol-omega alkanol chain derivative in muscle atrophy.

Muscular atrophy, a physiopathologic process associated with severe human diseases such as amyotrophic lateral sclerosis (ALS) or cancer, has been linked to reactive oxygen species (ROS) production. The Notch pathway plays a role in muscle development and in muscle regeneration upon physical injury. In this study, we explored the possibility that the Notch pathway participates in the ROS-related muscular atrophy occurring in cancer-associated cachexia and ALS. We also tested whether hybrid compounds of tocopherol, harboring antioxidant activity, and the omega-alkanol chain, presenting cytoprotective activity, might reduce muscle atrophy and impact the Notch pathway. We identified one tocopherol-omega alkanol chain derivative, AGT251, protecting myoblastic cells against known cytotoxic agents. We showed that this compound presenting antioxidant activity counteracts the induction of the Notch pathway by cytotoxic stress, leading to a decrease of Notch1 and Notch3 expression. At the functional level, these regulations correlated with a repression of the Notch target gene Hes1 and the atrophy/remodeling gene MuRF1. Importantly, we also observed an induction of Notch3 and Hes1 expression in two murine models of muscle atrophy: a doxorubicin-induced cachexia model and an ALS murine model expressing mutated superoxide dismutase 1. In both models, the induction of Notch3 and Hes1 were partially opposed by AGT251, which correlated with ameliorations in body and muscle weight, reduction of muscular atrophy markers, and improved survival. Altogether, we identified a compound of the tocopherol family that protects against muscle atrophy in various models, possibly through the regulation of the Notch pathway.

Conditional deletion of neurogenin-3 using Nkx2.1iCre results in a mouse model for the central control of feeding, activity and obesity.

The ventral hypothalamus acts to integrate visceral and systemic information to control energy balance. The basic helix-loop-helix transcription factor neurogenin-3 (Ngn3) is required for pancreatic ß-cell development and has been implicated in neuronal development in the hypothalamus. Here, we demonstrate that early embryonic hypothalamic inactivation of Ngn3 (also known as Neurog3) in mice results in rapid post-weaning obesity that is associated with hyperphagia and reduced energy expenditure. This obesity is caused by loss of expression of Pomc in Pomc- and Cart-expressing (Pomc/Cart) neurons in the arcuate nucleus, indicating an incomplete specification of anorexigenic first order neurons. Furthermore, following the onset of obesity, both the arcuate and ventromedial hypothalamic nuclei become insensitive to peripheral leptin treatment. This conditional mouse mutant therefore represents a novel model system for obesity that is associated with hyperphagia and underactivity, and sheds new light upon the roles of Ngn3 in the specification of hypothalamic neurons controlling energy balance.

Induction of caspase 8 and reactive oxygen species by ruthenium-derived anticancer compounds with improved water solubility and cytotoxicity.

Organometallic compounds which contain metals, such as ruthenium or gold, have been investigated as a replacement for platinum-derived anticancer drugs. They often show good antitumor effects, but the identification of their precise mode of action or their pharmacological optimization is still challenging. We have previously described a class of ruthenium(II) compounds with interesting anticancer properties. In comparison to cisplatin, these molecules have lower side effects, a reduced ability to interact with DNA, and they induce cell death in absence of p53 through CHOP/DDIT3. We have now optimized these molecules by improving their cytotoxicity and their water solubility. In this article, we demonstrate that by changing the ligands around the ruthenium we modify the ability of the compounds to interact with DNA. We show that these optimized molecules reduce tumor growth in different mouse models and retain their ability to induce CHOP/DDIT3. However, they are more potent inducers of cancer cell death and trigger the production of reactive oxygen species and the activation of caspase 8. More importantly, we show that blocking reactive oxygen species production or caspase 8 activity reduces significantly the activity of the compounds. Altogether our data suggest that water-soluble ruthenium(II)-derived compounds represent an interesting class of molecules that, depending on their structures, can target several pro-apoptotic signaling pathways leading to reactive oxygen species production and caspase 8 activation.

Complex regulation of p73 isoforms after alteration of amyloid precursor polypeptide (APP) function and DNA damage in neurons.

Genetic ablations of p73 have shown its implication in the development of the nervous system. However, the relative contribution of ΔNp73 and TAp73 isoforms in neuronal functions is still unclear. In this study, we have analyzed the expression of these isoforms during neuronal death induced by alteration of the amyloid-ß precursor protein function or cisplatin. We observed a concomitant up-regulation of a TAp73 isoform and a down-regulation of a ΔNp73 isoform. The shift in favor of the pro-apoptotic isoform correlated with an induction of the p53/p73 target genes such as Noxa. At a functional level, we showed that TAp73 induced neuronal death and that ΔNp73 has a neuroprotective role toward amyloid-ß precursor protein alteration or cisplatin. We investigated the mechanisms of p73 expression and found that the TAp73 expression was regulated at the promoter level. In contrast, regulation of ΔNp73 protein levels was regulated by phosphorylation at residue 86 and multiple proteases. Thus, this study indicates that tight transcriptional and post-translational mechanisms regulate the p73 isoform ratios that play an important role in neuronal survival.