Iron-Sensitive Prodrugs That Trigger Active Ferroptosis in Drug-Tolerant Pancreatic Cancer Cells.

Persister cancer cells represent rare populations of cells resistant to therapy. Cancer cells can exploit epithelial-mesenchymal plasticity to adopt a drug-tolerant state that does not depend on genetic alterations. Small molecules that can interfere with cell plasticity or kill cells in a cell state-dependent manner are highly sought after. Salinomycin has been shown to kill cancer cells in the mesenchymal state by sequestering iron in lysosomes, taking advantage of the iron addiction of this cell state. Here, we report the chemo- and stereoselective synthesis of a series of structurally complex small molecule chimeras of salinomycin derivatives and the iron-reactive dihydroartemisinin. We show that these chimeras accumulate in lysosomes and can react with iron to release bioactive species, thereby inducing ferroptosis in drug-tolerant pancreatic cancer cells and biopsy-derived organoids of pancreatic ductal adenocarcinoma. This work paves the way toward the development of new cancer medicines acting through active ferroptosis.

Combating pancreatic cancer chemoresistance by triggering multiple cell death pathways.

Pancreatic cancer is the fourth most common cause of cancer-associated death in western countries, where the incidence and number of deaths are increasing every year. Intrinsic or acquired resistance of tumor cells to chemotherapy agents is the major reason for failure of traditional cancer treatment. Several factors are implicated in this impressive resistance; however, of these, it is important to highlight the extensive cellular heterogeneity of these tumors. This heterogeneity is linked to a wide range of sensitivity that different clones in the same tumor display to chemotherapeutic agents. Accordingly, recent findings in this field have discovered new therapeutic targets in order to develop new combinatory treatments, as well as to induce several cell death pathways and reduce therapy-threshold and likelihood of future resistance. Accordingly, recent research has focused on targeting mitochondria, an organelle with key roles regulating cell death signaling pathways, such as apoptosis, necroptosis, autophagy, ferroptosis, or parthanatos. These findings - identifying new compounds, alone or in combination, that can target pancreatic ductal adenocarcinoma cell resistance - could be the key to future treatments.

Targeting intrinsically disordered proteins involved in cancer.

Intrinsically disordered proteins (IDPs) do not have a well-defined structure under physiological conditions, but they have key roles in cell signaling and regulation, and they are frequently related to the development of diseases, such as cancer and other malignancies. This has converted IDPs in attractive therapeutic targets; however, targeting IDPs is challenging because of their dynamic nature. In the last years, different experimental and computational approaches, as well as the combination of both, have been explored to identify molecules to target either the hot-spots or the allosteric sites of IDPs. In this review, we summarize recent developments in successful targeting of IDPs, all of which are involved in different cancer types. The strategies used to develop and design (or in one particular example, to repurpose) small molecules targeting IDPs are, in a global sense, similar to those used in well-folded proteins: (1) screening of chemically diverse or target-oriented compound libraries; or (2) study of the interfaces involved in recognition of their natural partners, and design of molecular candidates capable of binding to such binding interface. We describe the outcomes of using these approaches in targeting IDPs involved in cancer, in the view to providing insight, to target IDPs in general. In a broad sense, the designed small molecules seem to target the most hydrophobic regions of the IDPs, hampering macromolecule (DNA or protein)-IDP interactions; furthermore, in most of the molecule-IDP complexes described so far, the protein remains disordered.

Targeting Fibrosis: The Bridge That Connects Pancreatitis and Pancreatic Cancer.

Pancreatic fibrosis is caused by the excessive deposits of extracellular matrix (ECM) and collagen fibers during repeated necrosis to repair damaged pancreatic tissue. Pancreatic fibrosis is frequently present in chronic pancreatitis (CP) and pancreatic cancer (PC). Clinically, pancreatic fibrosis is a pathological feature of pancreatitis and pancreatic cancer. However, many new studies have found that pancreatic fibrosis is involved in the transformation from pancreatitis to pancreatic cancer. Thus, the role of fibrosis in the crosstalk between pancreatitis and pancreatic cancer is critical and still elusive; therefore, it deserves more attention. Here, we review the development of pancreatic fibrosis in inflammation and cancer, and we discuss the therapeutic strategies for alleviating pancreatic fibrosis. We further propose that cellular stress response might be a key driver that links fibrosis to cancer initiation and progression. Therefore, targeting stress proteins, such as nuclear protein 1 (NUPR1), could be an interesting strategy for pancreatic fibrosis and PC treatment.

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B.

Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 µM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.

Dendrimers as Competitors of Protein-Protein Interactions of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1.

NUPR1 is a protumoral multifunctional intrinsically disordered protein, which is activated during the acute phases of pancreatitis, interacting with several biomolecules through residues around Ala33 and Thr68. Because of the large size of this hot-spot, designed small molecules could be insufficient to modulate all NUPR1 functions. In this work, we studied NUPR1 interactions with dendrimers by using biophysical techniques and in silico methods. Our results, obtained with different functionalized dendrimers (anionic, cationic and neutral) and several of their generations, indicate that NUPR1 was bound to the dendrimers. Functionalities at the dendrimer periphery modulated the affinity for NUPR1, and for any dendrimer, the affinity increased with generation. The affinities of most of the dendrimers were in the range 4-40 × 103 M-1, and those of the [Gn]-PhCO2Na dendrimers were similar to those of NUPR1 for its natural partners (0.1-1 × 106 M-1). In all dendrimers, the residues of NUPR1 first affected upon binding were located around Ala33, indicating that NUPR1 employs the same hot-spot to recognize any natural or synthetic molecule.

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca2+ concentration ([Ca2+ ]c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca2+ ]c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells.

Amphipathic helical peptides hamper protein-protein interactions of the intrinsically disordered chromatin nuclear protein 1 (NUPR1).

BACKGROUND: NUPR1 is a multifunctional intrinsically disordered protein (IDP) involved, among other functions, in chromatin remodelling, and development of pancreatic ductal adenocarcinoma (PDAC). It interacts with several biomolecules through hydrophobic patches around residues Ala33 and Thr68. The drug trifluoperazine (TFP), which hampers PDAC development in xenografted mice, also binds to those regions. Because of the large size of the hot-spot interface of NUPR1, small molecules could not be adequate to modulate its functions. METHODS: We explored how amphipathic helical-designed peptides were capable of interacting with wild-type NUPR1 and the Thr68Gln mutant, inhibiting the interaction with NUPR1 protein partners. We used in vitro biophysical techniques (fluorescence, circular dichroism (CD), nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC)), in silico studies (docking and molecular dynamics (MD)), and in cellulo protein ligation assays (PLAs) to study the interaction. RESULTS: Peptide dissociation constants towards wild-type NUPR1 were ~ 3 µM, whereas no interaction was observed with the Thr68Gln mutant. Peptides interacted with wild-type NUPR1 residues around Ala33 and residues at the C terminus, as shown by NMR. The computational results clarified the main determinants of the interactions, providing a mechanism for the ligand-capture that explains why peptide binding was not observed for Thr68Gln mutant. Finally, the in cellulo assays indicated that two out of four peptides inhibited the interaction of NUPR1 with the C-terminal region of the Polycomb RING protein 1 (C-RING1B). CONCLUSIONS: Designed peptides can be used as lead compounds to inhibit NUPR1 interactions. GENERAL SIGNIFICANCE: Peptides may be exploited as drugs to target IDPs.

α-lipoic acid reduces postreperfusion syndrome in human liver transplantation - a pilot study.

A double-blind randomized controlled trial was performed to compare the safety and efficacy of α-lipoic acid (ALA) in liver transplantation (LT). The grafts were randomized to receive ALA or placebo before the cold ischemia time. Furthermore, patients transplanted with the ALA-perfused graft received 600 mg of intravenous ALA, while patients with the nonperfused graft received the placebo just before graft reperfusion. Hepatic biopsy was performed 2 h postreperfusion. Blood samples were collected before, during and 1 and 2 days after reperfusion. Quantitative polymerase chain reaction (qPCR) analysis was performed on biopsies to assess genes involved in the response to hypoxia, apoptosis, cell growth, survival and proliferation, cytokine production and tissue damage protection. Nine of 40 patients developed postreperfusion syndrome (PRS), but seven of them belonged to the control group. There was a decrease in PHD2 and an increase in alpha subunit of hypoxia-inducible factor-1 (HIF-1α) and baculoviral IAP repeat containing 2 (Birc2) transcript levels in the biopsies from the ALA-treated versus the control group of patients. Additionally, plasma levels of alarmins were lower in ALA-treated patients than control patients, which suggests that ALA-treated grafts are less inflammatory than untreated grafts. These results showed that ALA is safe for use in LT, induces gene changes that protect against hypoxia and oxidative stress and reduces the appearance of PRS.

Melatonin induces calcium mobilization and influences cell proliferation independently of MT1/MT2 receptor activation in rat pancreatic stellate cells.

Melatonin, the product of the pineal gland, possesses antioxidant, anti-inflammatory, and antitumor properties in different tissues, in addition to its role as regulator of biological rhythms. In this study, the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSCs) have been examined. Cell viability was studied using AlamarBlue® test. Cell-type specific markers and total amylase content were analyzed by immunocytochemistry and colorimetric methods, respectively. Changes in intracellular free Ca(2+) concentration were followed by fluorimetric analysis of fura-2-loaded cells. The cellular red-ox state was monitored following CM-H2DCFDA-derived fluorescence. Determination of the activation of p44/42 mitogen-activated protein kinase (MAPK), SAPK/JNK and p38 was measured by Western blot analysis. Our results show that PSCs viability decreased in the presence of 100 µM or 1 mM melatonin. However, in the presence of 1 or 10 µM melatonin, no changes in cell viability were observed. Melatonin MT1 and MT2 receptors could not be detected. Melatonin induced Ca(2+) mobilization from intracellular pools. In the presence of melatonin, activation of crucial components of MAPKs pathway was noticed. Finally, the indole did not change the oxidative state of PSCs, but exerted a protective effect against H2O2-induced oxidation. We conclude that melatonin, at pharmacological concentrations, might regulate cellular proliferation of PSCs independently of specific plasma membrane receptors.

Pharmacological dose of melatonin reduces cytosolic calcium load in response to cholecystokinin in mouse pancreatic acinar cells.

Intracellular Ca(2+) overload has been considered a common pathological precursor of pancreatic injury. In this study, the effects of melatonin on Ca(2+) mobilization induced by cholecystokinin octapeptide (CCK-8) in freshly isolated mouse pancreatic acinar cells have been examined. Changes in intracellular free Ca(2+) concentration were followed by single cell fluorimetry. For this purpose, cells were loaded with the Ca(2+)-sensitive fluorescent dye fura-2-acetoxymethyl ester. In order to evaluate the contribution of Ca(2+) transport at the plasma membrane, at the endoplasmic reticulum (ER) or at the mitochondria, cells were incubated with CCK-8 alone or in combination with LaCl3, thapsigargin (Tps), or FCCP to, respectively, uncouple Ca(2+) transport at these localizations. The experiments were performed in the absence or in the presence of melatonin in combination with the stimuli mentioned. Our results show that the total Ca(2+) mobilization evoked by CCK-8 was attenuated by a 30% in the presence of 100 µM melatonin compared with the responses induced by CCK-8 alone. Upon inhibition of Ca(2+) transport into the ER by Tps, Ca(2+) mobilization was also reduced in the presence of melatonin. In the presence of LaCl3 plus melatonin, the total Ca(2+) mobilization induced by CCK-8 was significantly decreased, compared with the response obtained without melatonin but in the presence of LaCl3. No major differences were found when the cells were incubated with CCK-8 or Tps alone or in combination with LaCl3 plus melatonin and FCCP, compared with the responses obtained in the absence of FCCP. The initial Ca(2+) release from intracellular stores evoked by CCK-8 or Tps was not significantly reduced in the presence of melatonin. The effect of melatonin could be explained on the basis of a stimulated Ca(2+) transport out of the cell through the plasma membrane and by a stimulation of Ca(2+) reuptake into the ER. Accumulation of Ca(2+) into mitochondria might not be a major mechanism stimulated by melatonin. We conclude that melatonin alleviates intracellular Ca(2+) accumulation, a situation potentially leading to cell damage in the exocrine pancreas.

Targeting NUPR1-dependent stress granules formation to induce synthetic lethality in KrasG12D-driven tumors.

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.

Stroma-targeting strategies in pancreatic cancer: a double-edged sword.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with limited treatment options and terrible long-term survival, and it is expected to become the second leading cause of cancer-related death by 2030. One reason why this cancer is so aggressive and resistant is the formation of dense stroma that surrounds the neoplastic epithelium, which promotes tumor progression, invasion, metastasis, and resistance. The three major components of PDAC stroma are extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), and vasculature. The dense ECM acts as a natural physical barrier, impeding drug penetration to PDAC tumor cells. Consequently, the method that combines stroma-targeting with anticancer therapy may be a viable alternative for increasing drug penetration. Additionally, blood vessels are key entities of the tumor stroma, serving as a pathway for nutrition as well as the only way for chemical medicines and immune cells to act. Finally, PDAC CAFs and tumor cells have crosstalk effects in the tumor microenvironment, where they are responsible for enhanced matrix deposition. In this review, we aim to provide an overview of our current comprehension of the three key components of PDAC stroma and the new promising therapeutic targets for PDAC.

Intrinsically Disordered Chromatin Protein NUPR1 Binds to the Enzyme PADI4.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 µM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.

Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells.

We have investigated the effects of melatonin on major pathways related with cellular proliferation and energetic metabolism in pancreatic stellate cells. In the presence of melatonin (1 mM, 100 µM, 10 µM, or 1 µM), decreases in the phosphorylation of c-Jun N-terminal kinase and of p44/42 and an increase in the phosphorylation of p38 were observed. Cell viability dropped in the presence of melatonin. A rise in the phosphorylation of AMP-activated protein kinase was detected in the presence of 1 mM and 100 µM melatonin. Treatment with 1 mM melatonin decreased the phosphorylation of protein kinase B, whereas 100 µM and 10 µM melatonin increased its phosphorylation. An increase in the generation of mitochondrial reactive oxygen species and a decrease of mitochondrial membrane potential were noted following melatonin treatment. Basal and maximal respiration, ATP production by oxidative phosphorylation, spare capacity, and proton leak dropped in the presence of melatonin. The expression of complex I of the mitochondrial respiratory chain was augmented in the presence of melatonin. Conversely, in the presence of 1 mM melatonin, decreases in the expression of mitofusins 1 and 2 were detected. The glycolysis and the glycolytic capacity were diminished in cells treated with 1 mM or 100 µM melatonin. Increases in the expression of phosphofructokinase-1 and lactate dehydrogenase were noted in cells incubated with 100 µM, 10 µM, or 1 µM melatonin. The expression of glucose transporter 1 was increased in cells incubated with 10 µM or 1 µM melatonin. Conversely, 1 mM melatonin decreased the expression of all three proteins. Our results suggest that melatonin, at pharmacological concentrations, might modulate mitochondrial physiology and energy metabolism in addition to major pathways involved in pancreatic stellate cell proliferation.

Resveratrol mobilizes Ca2+ from intracellular stores and induces c-Jun N-terminal kinase activation in tumoral AR42J cells.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin naturally found in grapes and red wine, is a redox-active compound endowed with significant positive activities. In this study, the effects of resveratrol on intracellular free Ca(2+) concentration ([Ca(2+)](c)) and on cell viability in tumoral AR42J pancreatic cells are examined. The results show that resveratrol (100 µM and 1 mM) induced changes in [Ca(2+)](c), that consisted of single or short lasting spikes followed by a slow reduction toward a value close to the resting level. Lower concentrations of resveratrol (1 and 10 µM) did not show detectable effects on [Ca(2+)](c). Depletion of intracellular Ca(2+) stores by stimulation of cells with 1 nM CCK-8, 20 pM CCK-8 or 1 µM thapsigargin, blocked Ca(2+) responses evoked by resveratrol. Conversely, prior stimulation of cells with resveratrol inhibited Ca(2+) mobilization in response to a secondary application of CCK-8 or thapsigargin. In addition, resveratrol inhibited oscillations in [Ca(2+)](c) evoked by a physiological concentration of CCK-8 (20 pM). On the other hand, incubation of cells in the presence of resveratrol induced a reduction of cell viability. Finally, incubation of AR42J cells in the presence of resveratrol led to activation of c-Jun N-terminal kinase (JNK), a mitogen-activated protein kinase responsive to stress stimuli. Activation of JNK was reduced in the absence of extracellular Ca(2+). In summary, the results show that resveratrol releases Ca(2+) from intracellular stores, most probably from the endoplasmic reticulum, and reduces AR42J cells viability. Reorganization of cell's survival/death processes in the presence of resveratrol may involve Ca(2+)-mediated JNK activation.

Melatonin modulates metabolic adaptation of pancreatic stellate cells subjected to hypoxia.

Pancreatic stellate cells (PSCs), the main cell type responsible for the development of fibrosis in pancreatic cancer, proliferate actively under hypoxia. Melatonin has received attention as a potential antifibrotic agent due to its anti-proliferative actions on PSCs. In this work, we investigated the activation of the PI3K/Akt/mTOR pathway and the metabolic adaptations that PSCs undergo under hypoxic conditions, as well as the probable modulation by melatonin. Incubation of cells under hypoxia induced an increase in cell proliferation, and in the expression of alpha-smooth muscle actin and of collagen type 1. In addition, an increase in the phosphorylation of Akt was observed, whereas a decrease in the phosphorylation of PTEN and GSK-3b was noted. The phosphorylation of mTOR and its substrate p70 S6K was decreased under hypoxia. Treatment of PSCs with melatonin under hypoxia diminished cell proliferation, the levels of alpha-smooth muscle actin and of collagen type 1, the phosphorylation of Akt and increased phosphorylation of mTOR. Mitochondrial activity decreased in PSCs under hypoxia. A glycolytic shift was observed. Melatonin further decreased mitochondrial activity. Under hypoxia, no increase in autophagic flux was noted. However, melatonin treatment induced autophagy activation. Nevertheless, inhibition of this process did not induce detectable changes in the viability of cells treated with melatonin. We conclude that PSCs undergo metabolic adaptation under hypoxia that might help them survive and that pharmacological concentrations of melatonin modulate cell responses to hypoxia. Our results contribute to the knowledge of the mechanisms by which melatonin could modulate fibrosis within the pancreas.

NUPR1 protects against hyperPARylation-dependent cell death.

Proteomic, cellular and biochemical analysis of the stress protein NUPR1 reveals that it binds to PARP1 into the nucleus and inhibits PARP1 activity in vitro. Mutations on residues Ala33 or Thr68 of NUPR1 or treatment with its inhibitor ZZW-115 inhibits this effect. PARylation induced by 5-fluorouracil (5-FU) treatment is strongly enhanced by ZZW-115 and associated with a decrease of NAD+/NADH ratio and rescued by the PARP inhibitor olaparib. Cell death induced by ZZW-115 treatment of pancreas cancer-derived cells is rescued by olaparib and improved with PARG inhibitor PDD00017273. The mitochondrial catastrophe induced by ZZW-115 treatment or by genetic inactivation of NUPR1 is associated to a hyperPARylation of the mitochondria, disorganization of the mitochondrial network, mitochondrial membrane potential decrease, and with increase of superoxide production, intracellular level of reactive oxygen species (ROS) and cytosolic levels of Ca2+. These features are rescued by olaparib or NAD+ precursor nicotinamide mononucleotide in a dose-dependent manner and partially by antioxidants treatments. In conclusion, inactivation of NUPR1 induces a hyperPARylation, which in turn, induces a mitochondrial catastrophe and consequently a cell death through a non-canonical Parthanatos, since apoptosis inducing-factor (AIF) is not translocated out of the mitochondria.

NUPR1: A Critical Regulator of the Antioxidant System.

Nuclear protein 1 (NUPR1) is a small intrinsically disordered protein (IDP) activated in response to various types of cellular stress, including endoplasmic reticulum (ER) stress and oxidative stress. Reactive oxygen species (ROS) are mainly produced during mitochondrial oxidative metabolism, and directly impact redox homeostasis and oxidative stress. Ferroptosis is a ROS-dependent programmed cell death driven by an iron-mediated redox reaction. Substantial evidence supports a maintenance role of the stress-inducible protein NUPR1 on cancer cell metabolism that confers chemotherapeutic resistance by upregulating mitochondrial function-associated genes and various antioxidant genes in cancer cells. NUPR1, identified as an antagonist of ferroptosis, plays an important role in redox reactions. This review summarizes the current knowledge on the mechanism behind the observed impact of NUPR1 on mitochondrial function, energy metabolism, iron metabolism, and the antioxidant system. The therapeutic potential of genetic or pharmacological inhibition of NUPR1 in cancer is also discussed. Understanding the role of NUPR1 in the antioxidant system and the mechanisms behind its regulation of ferroptosis may promote the development of more efficacious strategies for cancer therapy.

NUPR1 inhibitor ZZW-115 induces ferroptosis in a mitochondria-dependent manner.

Ferroptosis is an iron-dependent cell death characterized by the accumulation of hydroperoxided phospholipids. Here, we report that the NUPR1 inhibitor ZZW-115 induces ROS accumulation followed by a ferroptotic cell death, which could be prevented by ferrostatin-1 (Fer-1) and ROS-scavenging agents. The ferroptotic activity can be improved by inhibiting antioxidant factors in pancreatic ductal adenocarcinoma (PDAC)- and hepatocellular carcinoma (HCC)-derived cells. In addition, ZZW-115-treatment increases the accumulation of hydroperoxided lipids in these cells. We also found that a loss of activity and strong deregulation of key enzymes involved in the GSH- and GPX-dependent antioxidant systems upon ZZW-115 treatment. These results have been validated in xenografts induced with PDAC- and HCC-derived cells in nude mice during the treatment with ZZW-115. More importantly, we demonstrate that ZZW-115-induced mitochondrial morphological changes, compatible with the ferroptotic process, as well as mitochondrial network disorganization and strong mitochondrial metabolic dysfunction, which are rescued by both Fer-1 and N-acetylcysteine (NAC). Of note, the expression of TFAM, a key regulator of mitochondrial biogenesis, is downregulated by ZZW-115. Forced expression of TFAM is able to rescue morphological and functional mitochondrial alterations, ROS production, and cell death induced by ZZW-115 or genetic inhibition of NUPR1. Altogether, these results demonstrate that the mitochondrial cell death mediated by NUPR1 inhibitor ZZW-115 is fully rescued by Fer-1 but also via TFAM complementation. In conclusion, TFAM could be considered as an antagonist of the ferroptotic cell death.