Tumor suppressor Par-4 activates autophagy-dependent ferroptosis.

Ferroptosis is a unique iron-dependent form of non-apoptotic cell death characterized by devastating lipid peroxidation. Whilst growing evidence suggests that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms regulating ferroptosis are largely unknown. In this study, through an unbiased RNA-sequencing screening, we demonstrate the activation of a multi-faceted tumor-suppressor protein Par-4/PAWR during ferroptosis. Functional studies reveal that genetic depletion of Par-4 effectively blocks ferroptosis, whereas Par-4 overexpression sensitizes cells to undergo ferroptosis. More importantly, we have determined that Par-4-triggered ferroptosis is mechanistically driven by the autophagic machinery. Upregulation of Par-4 promotes activation of ferritinophagy (autophagic degradation of ferritin) via the nuclear receptor co-activator 4 (NCOA4), resulting in excessive release of free labile iron and, hence, enhanced lipid peroxidation and ferroptosis. Inhibition of Par-4 dramatically suppresses the NCOA4-mediated ferritinophagy signaling axis. Our results also establish that Par-4 activation positively correlates with reactive oxygen species (ROS) production, which is critical for ferritinophagy-mediated ferroptosis. Furthermore, Par-4 knockdown effectively blocked ferroptosis-mediated tumor suppression in the mouse xenograft models. Collectively, these findings reveal that Par-4 has a crucial role in ferroptosis, which could be further exploited for cancer therapy.

Positive regulation of oxidative phosphorylation by nuclear myosin 1 protects cells from metabolic reprogramming and tumorigenesis in mice.

Metabolic reprogramming is one of the hallmarks of tumorigenesis. Here, we show that nuclear myosin 1 (NM1) serves as a key regulator of cellular metabolism. NM1 directly affects mitochondrial oxidative phosphorylation (OXPHOS) by regulating mitochondrial transcription factors TFAM and PGC1α, and its deletion leads to underdeveloped mitochondria inner cristae and mitochondrial redistribution within the cell. These changes are associated with reduced OXPHOS gene expression, decreased mitochondrial DNA copy number, and deregulated mitochondrial dynamics, which lead to metabolic reprogramming of NM1 KO cells from OXPHOS to aerobic glycolysis.This, in turn, is associated with a metabolomic profile typical for cancer cells, namely increased amino acid-, fatty acid-, and sugar metabolism, and increased glucose uptake, lactate production, and intracellular acidity. NM1 KO cells form solid tumors in a mouse model, suggesting that the metabolic switch towards aerobic glycolysis provides a sufficient carcinogenic signal. We suggest that NM1 plays a role as a tumor suppressor and that NM1 depletion may contribute to the Warburg effect at the onset of tumorigenesis.

pH-Responsive Upconversion Mesoporous Silica Nanospheres for Combined Multimodal Diagnostic Imaging and Targeted Photodynamic and Photothermal Cancer Therapy.

Photodynamic therapy (PDT) and photothermal therapy (PTT) have gained considerable attention as potential alternatives to conventional cancer treatments. However, these approaches remain limited by low solubility, poor stability, and inefficient targeting of many common photosensitizers (PSs) and photothermal agents (PTAs). To overcome the aforementioned limitations, we engineered biocompatible and biodegradable tumor-targeted upconversion nanospheres with imaging capabilities. The multifunctional nanospheres consist of a sodium yttrium fluoride core doped with lanthanides (ytterbium, erbium, and gadolinium) and the PTA bismuth selenide (NaYF4:Yb/Er/Gd,Bi2Se3) enveloped in a mesoporous silica shell that encapsulates a PS, chlorin e6 (Ce6), within its pores. NaYF4:Yb/Er converts deeply penetrating near-infrared (NIR) light to visible light, which excites Ce6 to generate cytotoxic reactive oxygen species (ROS), while Bi2Se3 efficiently converts absorbed NIR light to heat. Additionally, Gd enables magnetic resonance imaging of the nanospheres. The mesoporous silica shell is coated with DPPC/cholesterol/DSPE-PEG to retain the encapsulated Ce6 and prevent serum protein adsorption and macrophage recognition that hinder tumor targeting. Finally, the coat is conjugated to the acidity-triggered rational membrane (ATRAM) peptide, which promotes specific and efficient internalization into malignant cells in the mildly acidic microenvironment of tumors. The nanospheres facilitated tumor magnetic resonance and thermal and fluorescence imaging and exhibited potent NIR laser light-induced anticancer effects in vitro and in vivo via combined ROS production and localized hyperthermia, with negligible toxicity to healthy tissue, hence markedly extending survival. Our results demonstrate that the ATRAM-functionalized, lipid/PEG-coated upconversion mesoporous silica nanospheres (ALUMSNs) offer multimodal diagnostic imaging and targeted combinatorial cancer therapy.

pH-responsive upconversion mesoporous silica nanospheres for combined multimodal diagnostic imaging and targeted photodynamic and photothermal cancer therapy.

Photodynamic therapy (PDT) and photothermal therapy (PTT) have garnered considerable interest as non-invasive cancer treatment modalities. However, these approaches remain limited by low solubility, poor stability and inefficient targeting of many common photosensitizers (PSs) and photothermal agents (PTAs). To overcome these limitations, we have designed biocompatible and biodegradable tumor-targeted upconversion nanospheres with imaging capabilities. The multifunctional nanospheres consist of a sodium yttrium fluoride core doped with lanthanides (ytterbium, erbium and gadolinium) and bismuth selenide (NaYF 4 :Yb/Er/Gd,Bi 2 Se 3 ) within a mesoporous silica shell that encapsulates a PS, Chlorin e6 (Ce6), in its pores. NaYF 4 :Yb/Er converts deeply penetrating near-infrared (NIR) light to visible light, which excites the Ce6 to generate cytotoxic reactive oxygen species (ROS), while the PTA Bi 2 Se 3 efficiently converts absorbed NIR light to heat. Additionally, Gd enables magnetic resonance imaging (MRI) of the nanospheres. The mesoporous silica shell is coated with lipid/polyethylene glycol (DPPC/cholesterol/DSPE-PEG) to ensure retention of the encapsulated Ce6 and minimize interactions with serum proteins and macrophages that impede tumor targeting. Finally, the coat is functionalized with the acidity-triggered rational membrane (ATRAM) peptide, which promotes specific and efficient internalization into cancer cells within the mildly acidic tumor microenvironment. Following uptake by cancer cells in vitro , NIR laser irradiation of the nanospheres caused substantial cytotoxicity due to ROS production and hyperthermia. The nanospheres facilitated tumor MRI and thermal imaging, and exhibited potent NIR laser light-induced antitumor effects in vivo via combined PDT and PTT, with no observable toxicity to healthy tissue, thereby substantially prolonging survival. Our results demonstrate that the ATRAM-functionalized, lipid/PEG-coated upconversion mesoporous silica nanospheres (ALUMSNs) offer multimodal diagnostic imaging and targeted combinatorial cancer therapy.

Erratum: The amyloid-inhibiting NCAM-PrP peptide targets Aß peptide aggregation in membrane-mimetic environments.

[This corrects the article DOI: 10.1016/j.isci.2021.102852.].

Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting N†Terminus Hot Segments and Attenuates Cytotoxicity.

Invited for the cover of this issue is the group of Prof. Hamilton at New York University. The image depicts how cucurbit[7]uril inhibits islet amyloid polypeptide self-assembly that rescues rat insulinoma cells (a pancreatic ß-cell model) from assembly-associated cytotoxicity. Read the full text of the article at 10.1002/chem.202200456.

Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting N Terminus Hot Segments and Attenuates Cytotoxicity.

Two "hot segments" within an islet amyloid polypeptide are responsible for its self-assembly, which in turn is linked to the decline of ß-cells in type 2 diabetes (T2D). A readily available water-soluble, macrocyclic host, cucurbit[7]uril (CB[7]), effectively inhibits islet amyloid polypeptide (IAPP) aggregation through ion-dipole and hydrophobic interactions with different residues of the monomeric peptide in its random-coil conformation. A HSQC NMR study shows that CB[7] likely modulates IAPP self-assembly by interacting with and masking major residues present in the "hot segments" at the N terminus. CB[7] also prevents the formation of toxic oligomers and inhibits seed-catalyzed fibril proliferation. Importantly, CB[7] recovers rat insulinoma cells (RIN-m) from IAPP-assembly associated cytotoxicity.

RIN4 homologs from important crop species differentially regulate the Arabidopsis NB-LRR immune receptor, RPS2.

KEY MESSAGE: RIN4 homologs from important crop species differ in their ability to prevent ectopic activity of the nucleotide binding-leucine rich repeat resistance protein, RPS2. Pathogens deploy virulence effectors to perturb host processes. Plants utilize intracellular resistance (R) proteins to recognize pathogen effectors either by direct interaction or indirectly via effector-mediated perturbations of host components. RPM1-INTERACTING PROTEIN4 (RIN4) is a plant immune regulator that mediates the indirect activation of multiple, independently evolved R-proteins by multiple, unrelated effector proteins. One of these, RPS2 (RESISTANT TO P. SYRINGAE2), is activated upon cleavage of Arabidopsis (At)RIN4 by the Pseudomonas syringae effector AvrRpt2. To gain insight into the AvrRpt2-RIN4-RPS2 defense-activation module, we compared the function of AtRIN4 with RIN4 homologs present in a diverse range of plant species. We selected seven homologs containing conserved features of AtRIN4, including two NOI (Nitrate induced) domains, each containing a predicted cleavage site for AvrRpt2, and a C-terminal palmitoylation site predicted to mediate membrane tethering of the proteins. Palmitoylation-mediated tethering of AtRIN4 to the plasma membrane and cleavage by AvrRpt2 are required for suppression and activation of RPS2, respectively. While all seven homologs are localized at the plasma membrane, only four suppress RPS2 when transiently expressed in Nicotiana benthamiana. All seven homologs are cleaved by AvrRpt2 and, for those homologs that are able to suppress RPS2, cleavage relieves suppression of RPS2. Further, we demonstrate that the membrane-tethered, C-terminal AvrRpt2-generated cleavage fragment is sufficient for the suppression of RPS2. Lastly, we show that the membrane localization of RPS2 is unaffected by its suppression or activation status.

The amyloid-inhibiting NCAM-PrP peptide targets Aß peptide aggregation in membrane-mimetic environments.

Substantial research efforts have gone into elucidating the role of protein misfolding and self-assembly in the onset and progression of Alzheimer's disease (AD). Aggregation of the Amyloid-ß (Aß) peptide into insoluble fibrils is closely associated with AD. Here, we use biophysical techniques to study a peptide-based approach to target Aß amyloid aggregation. A peptide construct, NCAM-PrP, consists of a largely hydrophobic signal sequence linked to a positively charged hexapeptide. The NCAM-PrP peptide inhibits Aß amyloid formation by forming aggregates which are unavailable for further amyloid aggregation. In a membrane-mimetic environment, Aß and NCAM-PrP form specific heterooligomeric complexes, which are of lower aggregation states compared to Aß homooligomers. The Aß:NCAM-PrP interaction appears to take place on different aggregation states depending on the absence or presence of a membrane-mimicking environment. These insights can be useful for the development of potential future therapeutic strategies targeting Aß at several aggregation states.

Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function.

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimer's disease and type II diabetes, identified a tripyridylamide, ADH-6, that abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53's transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment effectively shrinks xenografts harboring mutant p53, while exhibiting no toxicity to healthy tissue, thereby substantially prolonging survival. This study demonstrates the successful application of a bona fide small-molecule amyloid inhibitor as a potent anticancer agent.

Peptidomimetic-Based Vesicles Inhibit Amyloid-ß Fibrillation and Attenuate Cytotoxicity.

An interruption in Aß homeostasis leads to the deposit of neurotoxic amyloid plaques and is associated with Alzheimer's disease. A supramolecular strategy based on the assembly of peptidomimetic agents into functional vesicles has been conceived for the simultaneous inhibition of Aß42 fibrillation and expedited clearance of Aß42 aggregates. Tris-pyrrolamide peptidomimetic, ADH-353, contains one hydrophobic N-butyl and two hydrophilic N-propylamine side chains and readily forms vesicles under physiological conditions. These vesicles completely rescue both mouse neuroblastoma N2a and human neuroblastoma SH-SY5Y cells from the cytotoxicity that follows from Aß42 misfolding likely in mitochondria. Biophysical studies, including confocal imaging, demonstrate the biocompatibility and selectivity of the approach toward this aberrant protein assembly in cellular milieu.

Hexokinase II-Derived Cell-Penetrating Peptide Mediates Delivery of MicroRNA Mimic for Cancer-Selective Cytotoxicity.

Cancer cells are often characterized by elevated levels of mitochondrion-bound hexokinase II (HKII), which facilitates their survival, proliferation, and metastasis. Here, we have designed a cancer-selective cell-penetrating peptide (CPP) by covalently coupling a short penetration-accelerating sequence (PAS) to the mitochondrial membrane-binding N-terminal 15 amino acids of HKII (pHK). PAS-pHK mediates efficient cellular uptake and cytosolic delivery of a synthetic mimic of miR-126, a tumor suppressor miRNA downregulated in many malignancies. Following uptake by breast cancer MCF-7 cells, the CPP-miRNA conjugate is distributed throughout the cytosol and shows strong colocalization with mitochondria, where PAS-pHK induces depolarization of mitochondrial membrane potential, inhibition of metabolic activities, depletion of intracellular ATP levels, release of cytochrome c, and, finally, apoptosis. Concomitantly, the miR-126 cargo synergistically enhances the anticancer effects of PAS-pHK. Importantly, the PAS-pHK-miR-126 conjugate is not toxic to noncancerous MCF-10A and HEK-93 cells. Our results demonstrate the potential of PAS-pHK-mediated delivery of miRNA mimics as a novel cancer-selective therapeutic strategy.

Combating Proteins with Proteins: Engineering Cell-Penetrating Peptide Antagonists of Amyloid-ß Aggregation and Associated Neurotoxicity.

A central event that underlies the etiology of Alzheimer's disease (AD) is the self-assembly of the amyloid-ß (Aß) peptide into aggregates termed amyloids. Increasing evidence implicates soluble prefibrillar Aß oligomers in the neurodegeneration and synaptic dysfunction in AD. Recently we introduced a new class of highly promising antagonists of Aß amyloidogenesis: designed cell-penetrating peptides (CPPs). These CPPs combine the attractive intrinsic properties of peptides (high target specificity and selectivity, biocompatibility, biodegradability, and ease and low cost of production) with potent therapeutic effects (inhibition of Aß oligomerization, fiber formation, and neurotoxicity) and highly efficient delivery (to target cells and subcellular organelles).

pH-responsive high stability polymeric nanoparticles for targeted delivery of anticancer therapeutics.

The practical application of nanoparticles (NPs) as chemotherapeutic drug delivery systems is often hampered by issues such as poor circulation stability and targeting inefficiency. Here, we have utilized a simple approach to prepare biocompatible and biodegradable pH-responsive hybrid NPs that overcome these issues. The NPs consist of a drug-loaded polylactic-co-glycolic acid (PLGA) core covalently 'wrapped' with a crosslinked bovine serum albumin (BSA) shell designed to minimize interactions with serum proteins and macrophages that inhibit target recognition. The shell is functionalized with the acidity-triggered rational membrane (ATRAM) peptide to facilitate internalization specifically into cancer cells within the acidic tumor microenvironment. Following uptake, the unique intracellular conditions of cancer cells degrade the NPs, thereby releasing the chemotherapeutic cargo. The drug-loaded NPs showed potent anticancer activity in vitro and in vivo while exhibiting no toxicity to healthy tissue. Our results demonstrate that the ATRAM-BSA-PLGA NPs are a promising targeted cancer drug delivery platform.

Supercritical CO2 Processing Generates Aqueous Cisplatin Solutions with Enhanced Cancer Specificity.

Cisplatin is a highly toxic material used clinically as a potent chemotherapeutic. While effective against some cancers, toxicity limits widespread use and low solubility confounds delivery. To formulate a better tolerated and more water-soluble form of cisplatin, we designed a rapid expansion of supercritical solutions (RESS) technique with supercritical carbon dioxide (sc-CO2) to collect nanoclusters of cisplatin embedded in dry ice, in a dual-stage collection vessel cooled to liquid nitrogen temperature. These nanoclusters were solubilized in deionized water and further concentrated (up to 51.3 mM) by a Rotovap process, yielding stable cisplatin solutions with solubility up to 15 × (w/w) greater than that of normal cisplatin. Extensive material characterizations of the solutions were carried out to determine any chemical and/or structural changes of the RESS-processed cisplatin. In vitro cytotoxicity studies of these aqueous solutions showed increased cell viability and early apoptosis compared to equivalent concentrations of standard cisplatin solutions. In vivo studies using zebrafish embryos revealed that standard cisplatin solutions were acutely toxic and caused death of rapidly proliferating cells compared to RESS-processed cisplatin, which were better tolerated with reduced general cell death. Increased water solubility and matched chemical identity of RESS-processed aqueous cisplatin solutions indicate the potential to open up novel drug-delivery routes, which is beneficial for new pharmaceutical design and development.

Sub-stoichiometric inhibition of IAPP aggregation: a peptidomimetic approach to anti-amyloid agents.

Membrane-catalysed misfolding of islet amyloid polypeptide is associated with the death of ß-cells in type II diabetes (T2D). Most active compounds so far reported require high doses for inhibition of membrane bound IAPP fibrillation. Here, we describe a naphthalimide-appended oligopyridylamide-based α-helical mimetic, DM 1, for targeting membrane bound IAPP. DM 1 completely inhibits the aggregation of IAPP at doses of 0.2 equivalents. DM 1 is also effective at similarly low doses for inhibition of seed-catalyzed secondary nucleation. An NMR based study demonstrates that DM 1 modulates IAPP self-assembly by stabilizing and/or perturbing the N-terminus helix conformation. DM 1 at substoichiometric doses rescues rat insulinoma cells from IAPP-mediated cytotoxicity. Most importantly, 0.2 equivalents of DM 1 disaggregate preformed oligomers and fibrils and can reverse cytotoxicity by modulating toxic preformed oligomers and fibrils of IAPP into non-toxic conformations.

Pro-Inflammatory S100A9 Protein Aggregation Promoted by NCAM1 Peptide Constructs.

Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from Aß peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid self-assembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.

Cancer targeting peptides.

Despite continuing advances in the development of biomacromolecules for therapeutic purposes, successful application of these often large and hydrophilic molecules has been hindered by their inability to efficiently traverse the cellular plasma membrane. In recent years, cell-penetrating peptides (CPPs) have received considerable attention as a promising class of delivery vectors due to their ability to mediate the efficient import of a large number of cargoes in vitro and in vivo. However, the lack of target specificity of CPPs remains a major obstacle to their clinical development. To address this issue, researchers have developed strategies in which chemotherapeutic drugs are conjugated to cancer targeting peptides (CTPs) that exploit the unique characteristics of the tumor microenvironment or cancer cells, thereby improving cancer cell specificity. This review highlights several of these strategies that are currently in use, and discusses how multi-component nanoparticles conjugated to CTPs can be designed to provide a more efficient cancer therapeutic delivery strategy.

Mechanistic insights into the pH-dependent membrane peptide ATRAM.

pH-responsive peptides are promising therapeutic molecules that can specifically target the plasma membrane in the acidified extracellular medium that bathes cells in tumors. We designed the acidity-triggered rational membrane (ATRAM) peptide to have a pH-responsive membrane interaction. At physiological pH, ATRAM binds to the membrane surface in a largely unstructured conformation, while in acidic conditions it inserts into lipid bilayers forming a transmembrane helix. However, the molecular mechanism ATRAM uses to target and insert into tumor cells remains poorly understood. Here, we determined that ATRAM inserts into cancer cells with a preferential membrane orientation, where the C-terminus of the peptide traverses the plasma membrane and explores the cytoplasm. Using biophysical techniques, we determined that the membrane interaction of ATRAM is contingent on the concentration of the peptide. Kinetic studies showed that membrane insertion occurs in at least three steps, where only the first step was affected by the membrane density of ATRAM. These observations, combined with membrane binding and leakage data, indicate that the interaction of ATRAM with lipid membranes is dependent on its oligomerization state. SPECT/CT imaging in mice revealed that ATRAM accumulates in the blood pool, where it has a prolonged circulation time (> 4 h). Since fast peptide clearance and degradation in circulation are major problems for clinical development, we studied the mechanism ATRAM uses to remain in the blood stream. Using binding and transfer assays, we determined that ATRAM binds reversibly to human serum albumin. We propose that ATRAM uses albumin as a carrier in the blood stream to evade clearance and proteolysis before interacting with the plasma membrane of cancer cells. We also show that ATRAM is able to be deliver liposomes to cells in a pH dependent way. Our data highlight the potential of ATRAM as a specific therapeutic agent for diseases that lead to acidic tissues, including cancer.