ASNA-1 oxidation induced by cisplatin exposure enhances its cytotoxicity by selectively perturbing tail anchored protein targeting

ABSTRACTCisplatin is a frontline cancer treatment, but intrinsic or acquired resistance is common. We previously showed that ASNA-1/TRC40 inactivation increases cisplatin sensitivity in mammalian cells and a Caenorhabditis elegans asna-1 knockdown model. ASNA-1 has conserved tail-anchored protein (TAP) targeting and insulin secretion functions. Here we examined the mechanism of ASNA-1 action. We show that ASNA-1 exists in two physiologically-responsive redox states with separable TAP-targeting and insulin secretion functions. Cisplatin-generated ROS targeted ASNA-1 oxidation, resulting in a selective targeting defect of an ASNA-1-dependent TAP. Increased ASNA-1 oxidation sensitized worms to cisplatin cytotoxicity. Mutants with a redox balance favoring oxidized ASNA-1 were cisplatin sensitive as null mutants by diverting ASNA-1 away from its TAP-targeting role and instead perturbing endoplasmic reticulum (ER) function. Mutations in the ASNA-1 receptor required for TAP insertion induced equivalent cisplatin sensitivity. We reveal a previously undescribed cellular dysfunction induced by cisplatin, identify a cisplatin target, and show that drug exposure causes TAP targeting-induced ER dysfunction. Therapeutic oxidation of ASNA-1 could be a clinically useful means to increase cisplatin sensitivity, reduce cytotoxic drug doses, and counteract cisplatin resistance.AUTHOR SUMMARYCisplatin is a very effective anti-cancer drug and is widely used as a frontline treatment. However, tumor resistance limits its use. Tumor re-sensitization would improve cancer treatment. ASNA-1/TRC40 knockdown in Caenorhabditis elegans and mammals results in cisplatin hypersensitivity, but the underlying mechanistic details are largely unknown. We show that in C. elegans ASNA-1 mutants, increased cisplatin killing is coupled with delocalization of a tail-anchored protein, SEC-61β, a membrane protein that should reach the ER and is instead mistargeted. Like its homologs, the reduced form of worm ASNA-1 is needed for targeting activity. Targeting is blocked upon ASNA-1 oxidation after cisplatin treatment, likely via reactive oxygen species (ROS) generated by cisplatin treatment. Nevertheless, the oxidized form of the protein can execute other functions like insulin secretion. We show also that mutants with high oxidized ASNA-1 levels are cisplatin sensitive. Additionally, cisplatin induced mistargeting strictly acts through ASNA-1 inactivation. Thus, we define a pathway from cisplatin exposure that targets protein (ASNA-1) inactivation, consequently leading to mis-targeting of proteins that need ASNA-1 for their maturation. This multi-step process provides vital information about likely proteins that can be targeted by drugs to enhance cisplatin mediated killing and improve chemotherapy.SUMMARY STATEMENTSensitizing tumors to cisplatin would be of considerable therapeutic benefit. Here we show a novel mechanism of cisplatin sensitization via oxidation of ASNA-1 in a Caenorhabditis elegans model.

Download Full-text

Alternative redox forms of ASNA-1 separate insulin signaling from tail-anchored protein targeting and cisplatin resistance in C. elegans

Scientific Reports ◽

10.1038/s41598-021-88085-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Dorota Raj ◽

Ola Billing ◽

Agnieszka Podraza-Farhanieh ◽

Bashar Kraish ◽

Oskar Hemmingsson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Insulin Secretion ◽

Protein Targeting ◽

Cisplatin Resistance ◽

Acquired Resistance ◽

Endoplasmic Reticulum Membrane ◽

C Elegans ◽

Cisplatin Sensitivity ◽

Tumor Environment ◽

The One

AbstractCisplatin is a frontline cancer therapeutic, but intrinsic or acquired resistance is common. We previously showed that cisplatin sensitivity can be achieved by inactivation of ASNA-1/TRC40 in mammalian cancer cells and in Caenorhabditis elegans. ASNA-1 has two more conserved functions: in promoting tail-anchored protein (TAP) targeting to the endoplasmic reticulum membrane and in promoting insulin secretion. However, the relation between its different functions has remained unknown. Here, we show that ASNA-1 exists in two redox states that promote TAP-targeting and insulin secretion separately. The reduced state is the one required for cisplatin resistance: an ASNA-1 point mutant, in which the protein preferentially was found in the oxidized state, was sensitive to cisplatin and defective for TAP targeting but had no insulin secretion defect. The same was true for mutants in wrb-1, which we identify as the C. elegans homolog of WRB, the ASNA1/TRC40 receptor. Finally, we uncover a previously unknown action of cisplatin induced reactive oxygen species: cisplatin induced ROS drives ASNA-1 into the oxidized form, and selectively prevents an ASNA-1-dependent TAP substrate from reaching the endoplasmic reticulum. Our work suggests that ASNA-1 acts as a redox-sensitive target for cisplatin cytotoxicity and that cisplatin resistance is likely mediated by ASNA-1-dependent TAP substrates. Treatments that promote an oxidizing tumor environment should be explored as possible means to combat cisplatin resistance.

Download Full-text

A technology platform to assess multiple cancer agents simultaneously within a patient’s tumor

Science Translational Medicine ◽

10.1126/scitranslmed.aaa7489 ◽

2015 ◽

Vol 7 (284) ◽

pp. 284ra58-284ra58 ◽

Cited By ~ 49

Author(s):

Richard A. Klinghoffer ◽

S. Bahram Bahrami ◽

Beryl A. Hatton ◽

Jason P. Frazier ◽

Alicia Moreno-Gonzalez ◽

...

Keyword(s):

Anticancer Agents ◽

Drug Exposure ◽

Fundamental Problem ◽

Feasibility Studies ◽

Cancer Drug ◽

Tumor Resistance ◽

Multiple Cancer ◽

Technology Platform

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

Download Full-text

Psychogenic Mass Syndrome in Anti-Cancer Drug Exposure

PsycEXTRA Dataset ◽

10.1037/e572992012-432 ◽

2010 ◽

Author(s):

N. Magnavita ◽

I. lavicoli ◽

V. Leso ◽

A. Bergamaschi

Keyword(s):

Drug Exposure ◽

Cancer Drug ◽

Anti Cancer

Download Full-text

Smart Nanoparticles for Chemo-Based Combinational Therapy

Pharmaceutics ◽

10.3390/pharmaceutics13060853 ◽

2021 ◽

Vol 13 (6) ◽

pp. 853

Author(s):

Binita Shrestha ◽

Lijun Wang ◽

Eric M. Brey ◽

Gabriela Romero Uribe ◽

Liang Tang

Keyword(s):

Cancer Treatment ◽

Cancer Biology ◽

Complex Disease ◽

Rapid Development ◽

Acquired Resistance ◽

Therapeutic Index ◽

Chemotherapeutic Agents ◽

Combinational Therapy ◽

Precision Therapy ◽

External Stimuli

Cancer is a heterogeneous and complex disease. Traditional cancer therapy is associated with low therapeutic index, acquired resistance, and various adverse effects. With the increasing understanding of cancer biology and technology advancements, more strategies have been exploited to optimize the therapeutic outcomes. The rapid development and application of nanomedicine have motivated this progress. Combinational regimen, for instance, has become an indispensable approach for effective cancer treatment, including the combination of chemotherapeutic agents, chemo-energy, chemo-gene, chemo-small molecules, and chemo-immunology. Additionally, smart nanoplatforms that respond to external stimuli (such as light, temperature, ultrasound, and magnetic field), and/or to internal stimuli (such as changes in pH, enzymes, hypoxia, and redox) have been extensively investigated to improve precision therapy. Smart nanoplatforms for combinational therapy have demonstrated the potential to be the next generation cancer treatment regimen. This review aims to highlight the recent advances in smart combinational therapy.

Download Full-text

DRUG REPOSITION OF NON-CANCER DRUGS FOR CANCER TREATMENTS VIA PHARMACOVIGILANCE APPROACH - REPURPOSING DRUGS IN ONCOLOGY

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i2.29523 ◽

2019 ◽

pp. 310-314 ◽

Cited By ~ 1

Author(s):

Mrugank Bhaskarkumar Parmar ◽

Shital Panchal

Keyword(s):

Statistical Analysis ◽

Cancer Treatment ◽

Drug Repositioning ◽

Adverse Event Reporting System ◽

Adverse Event Reporting ◽

Cancer Drug ◽

Cancer Drugs ◽

Management Activity ◽

Post Marketing Surveillance ◽

Source Data

This study for drug repositioning has been performed for the drugs which are in the market since more than a decade and they are approved with their well-established efficacy and safety in human being. Objective of this study was to reposition the existing non-cancer drug therapy for cancer treatment, which is having well characterized pharmacologic profile with more efficacy and least toxicity as anti-neoplastic agent. We have retrieved the source data from FDA Adverse Event Reporting System (FAERS) for the last 13 years covering duration from 2004 to 2016 and analysed those using pharmacovigilance approach â€˜a proposed future novel pharmaceutical tool for drug repositionâ€™. Signal management activity was performed for statistical analysis. Result of statistical analysis derived that propranolol; metformin; pioglitazone; dabigatran and nitroglycerin are the existing non-cancer drugs which deserved for their direct / indirect reposition for cancer treatment and anti-neoplastic activity. Further studies retrieving the source data from other regulatory database (e.g. Eudravigilance of EMA and VigiFlow of WHO) and post-marketing surveillance study with the same objective may adjuvant our results for the reposition of existing drugs by pharmacovigilance approach.

Download Full-text

DDRE-07. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL INHIBITION

Neuro-Oncology ◽

10.1093/neuonc/noaa215.252 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii62-ii62

Author(s):

Elisa Izquierdo ◽

Diana Carvalho ◽

Alan Mackay ◽

Sara Temelso ◽

Jessica K R Boult ◽

...

Keyword(s):

Drug Exposure ◽

Mapk Pathway ◽

Synergistic Effects ◽

Acquired Resistance ◽

Mek Inhibitor ◽

Resistance Mechanisms ◽

Genetic Alterations ◽

Mesenchymal Transition

Abstract The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples, which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

Download Full-text

Caenorhabditis elegans SMG-2 Selectively Marks mRNAs Containing Premature Translation Termination Codons

Molecular and Cellular Biology ◽

10.1128/mcb.00410-07 ◽

2007 ◽

Vol 27 (16) ◽

pp. 5630-5638 ◽

Cited By ~ 39

Author(s):

Lisa Johns ◽

Andrew Grimson ◽

Sherry L. Kuchma ◽

Carrie Loushin Newman ◽

Philip Anderson

Keyword(s):

Caenorhabditis Elegans ◽

Mammalian Cells ◽

Translation Termination ◽

Yeast Two Hybrid ◽

Eukaryotic Mrnas ◽

Nonsense Mediated Mrna Decay ◽

Endogenous Genes ◽

Alternatively Spliced ◽

Two Hybrid ◽

Premature Translation Termination

ABSTRACT Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed “nonsense-mediated mRNA decay” (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with (“marks”) those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.

Download Full-text

Oxidative protein folding in the mammalian endoplasmic reticulum

Biochemical Society Transactions ◽

10.1042/bst0320655 ◽

2004 ◽

Vol 32 (5) ◽

pp. 655-658 ◽

Cited By ~ 51

Author(s):

C.E. Jessop ◽

S. Chakravarthi ◽

R.H. Watkins ◽

N.J. Bulleid

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Redox State ◽

Structure And Function ◽

Reduced Form ◽

Secretory Proteins ◽

Oxidative Protein Folding ◽

Disulphide Bonds ◽

And Function ◽

Substrate Proteins

Native disulphide bonds are essential for the structure and function of many membrane and secretory proteins. Disulphide bonds are formed, reduced and isomerized in the endoplasmic reticulum of mammalian cells by a family of oxidoreductases, which includes protein disulphide isomerase (PDI), ERp57, ERp72, P5 and PDIR. This review will discuss how these enzymes are maintained in either an oxidized redox state that allows them to form disulphide bonds in substrate proteins or a reduced form that allows them to perform isomerization and reduction reactions, how these opposing pathways may co-exist within the same compartment and why so many oxidoreductases exist when PDI alone can perform all three of these functions.

Download Full-text

Two Alternative Mechanisms That Regulate the Presentation of Apoptotic Cell Engulfment Signal in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e07-02-0138 ◽

2007 ◽

Vol 18 (8) ◽

pp. 3180-3192 ◽

Cited By ~ 73

Author(s):

Victor Venegas ◽

Zheng Zhou

Keyword(s):

Caenorhabditis Elegans ◽

Abc Transporter ◽

Germ Cells ◽

Mammalian Cells ◽

Developmental Stages ◽

Apoptotic Cell ◽

Somatic Cells ◽

Apoptotic Cells ◽

Phospholipid Scramblase ◽

Cell Engulfment

Phosphatidylserine exposed on the surface of apoptotic mammalian cells is considered an “eat-me” signal that attracts phagocytes. The generality of using phosphatidylserine as a clearance signal for apoptotic cells in animals and the regulation of this event remain uncertain. Using ectopically expressed mouse MFG-E8, a secreted phosphatidylserine-binding protein, we detected specific exposure of phosphatidylserine on the surface of apoptotic cells in Caenorhabditis elegans. Masking the surface phosphatidylserine inhibits apoptotic cell engulfment. CED-7, an ATP-binding cassette (ABC) transporter, is necessary for the efficient exposure of phosphatidylserine on apoptotic somatic cells, and for the recognition of these cells by phagocytic receptor CED-1. Alternatively, phosphatidylserine exposure on apoptotic germ cells is not CED-7 dependent, but instead requires phospholipid scramblase PLSC-1, a homologue of mammalian phospholipid scramblases. Moreover, deleting plsc-1 results in the accumulation of apoptotic germ cells but not apoptotic somatic cells. These observations suggest that phosphatidylserine might be recognized by CED-1 and act as a conserved eat-me signal from nematodes to mammals. Furthermore, the two different biochemical activities used in somatic cells (ABC transporter) and germ cells (phospholipid scramblase) suggest an increased complexity in the regulation of phosphatidylserine presentation in response to apoptotic signals in different tissues and during different developmental stages.

Download Full-text