Publications by authors named "Zdenek Skrott"

7 Publications

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Microthermal-induced subcellular-targeted protein damage in cells on plasmonic nanosilver-modified surfaces evokes a two-phase HSP-p97/VCP response.

Nat Commun 2021 01 29;12(1):713. Epub 2021 Jan 29.

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

Despite proteotoxic stress and heat shock being implicated in diverse pathologies, currently no methodology to inflict defined, subcellular thermal damage exists. Here, we present such a single-cell method compatible with laser-scanning microscopes, adopting the plasmon resonance principle. Dose-defined heat causes protein damage in subcellular compartments, rapid heat-shock chaperone recruitment, and ensuing engagement of the ubiquitin-proteasome system, providing unprecedented insights into the spatiotemporal response to thermal damage relevant for degenerative diseases, with broad applicability in biomedicine. Using this versatile method, we discover that HSP70 chaperone and its interactors are recruited to sites of thermally damaged proteins within seconds, and we report here mechanistically important determinants of such HSP70 recruitment. Finally, we demonstrate a so-far unsuspected involvement of p97(VCP) translocase in the processing of heat-damaged proteins. Overall, we report an approach to inflict targeted thermal protein damage and its application to elucidate cellular stress-response pathways that are emerging as promising therapeutic targets.
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http://dx.doi.org/10.1038/s41467-021-20989-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846584PMC
January 2021

Targeting the NPL4 Adaptor of p97/VCP Segregase by Disulfiram as an Emerging Cancer Vulnerability Evokes Replication Stress and DNA Damage while Silencing the ATR Pathway.

Cells 2020 02 18;9(2). Epub 2020 Feb 18.

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 77 147 Olomouc, Czech Republic.

Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.
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http://dx.doi.org/10.3390/cells9020469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072750PMC
February 2020

Disulfiram's anti-cancer activity reflects targeting NPL4, not inhibition of aldehyde dehydrogenase.

Oncogene 2019 10 7;38(40):6711-6722. Epub 2019 Aug 7.

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

Aldehyde dehydrogenase (ALDH) is a proposed biomarker and possible target to eradicate cancer stem cells. ALDH inhibition as a treatment approach is supported by anti-cancer effects of the alcohol-abuse drug disulfiram (DSF, Antabuse). Given that metabolic products of DSF, rather than DSF itself inhibit ALDH in vivo, and that DSF's anti-cancer activity is potentiated by copper led us to investigate the relevance of ALDH as the suggested molecular cancer-relevant target of DSF. Here we show that DSF does not directly inhibit ALDH activity in diverse human cell types, while DSF's in vivo metabolite, S-methyl-N,N-diethylthiocarbamate-sulfoxide inhibits ALDH activity yet does not impair cancer cell viability. Our data indicate that the anti-cancer activity of DSF does not involve ALDH inhibition, and rather reflects the impact of DSF's copper-containing metabolite (CuET), that forms spontaneously in vivo and in cell culture media, and kills cells through aggregation of NPL4, a subunit of the p97/VCP segregase. We also show that the CuET-mediated, rather than any ALDH-inhibitory activity of DSF underlies the preferential cytotoxicity of DSF towards BRCA1- and BRCA2-deficient cells. These findings provide evidence clarifying the confusing literature about the anti-cancer mechanism of DSF, a drug currently tested in clinical trials for repositioning in oncology.
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http://dx.doi.org/10.1038/s41388-019-0915-2DOI Listing
October 2019

Targeting genotoxic and proteotoxic stress-response pathways in human prostate cancer by clinically available PARP inhibitors, vorinostat and disulfiram.

Prostate 2019 03 29;79(4):352-362. Epub 2018 Nov 29.

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

Background: Castration-resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically-supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol-abuse drug Antabuse) and its copper-chelating metabolite CuET that inhibit protein turnover.

Methods: Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio-resistant stem-cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting.

Results: We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR-mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio-resistance of stem-cell like DU145-derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho-eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti-apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR-induced toxicity, yielding synergistic effects of CuET and YM155.

Conclusions: We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.
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http://dx.doi.org/10.1002/pros.23741DOI Listing
March 2019

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.

Nature 2017 12 6;552(7684):194-199. Epub 2017 Dec 6.

Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.
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http://dx.doi.org/10.1038/nature25016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730499PMC
December 2017

Linking the activity of bortezomib in multiple myeloma and autoimmune diseases.

Crit Rev Oncol Hematol 2014 Nov 10;92(2):61-70. Epub 2014 May 10.

Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic. Electronic address:

Since their introduction to the clinic 10 years ago, proteasome inhibitors have become the cornerstone of anti-multiple myeloma therapy. Despite significant progress in understanding the consequences of proteasome inhibition, the unique activity of bortezomib is still unclear. Disappointing results from clinical trials with bortezomib in other malignancies raise the question of what makes multiple myeloma so sensitive to proteasome inhibition. Successful administration of bortezomib in various immunological disorders that exhibit high antibody production suggests that the balance between protein synthesis and degradation is a key determinant of sensitivity to proteasome inhibition because a high rate of protein production is a shared characteristic in plasma and myeloma cells. Initial or acquired resistance to bortezomib remains a major obstacle in the clinic as in vitro data from cell lines suggest a key role for the β5 subunit mutation in resistance; however the mutation was not found in patient samples. Recent studies indicate the importance of selecting for a subpopulation of cells that produce lower amounts of paraprotein during bortezomib therapy.
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http://dx.doi.org/10.1016/j.critrevonc.2014.05.003DOI Listing
November 2014

Diethyldithiocarbamate complex with copper: the mechanism of action in cancer cells.

Mini Rev Med Chem 2012 Oct;12(12):1184-92

Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic.

The idea of "repurposing" of existing drugs provides an effective way to develop and identify new therapies. Disulfiram (Antabuse), a drug commonly used for the treatment of alcoholism, shows promising anticancer activity in both preclinical and clinical studies. In the human body, disulfiram is rapidly converted to its reduced metabolite, diethyldithiocarbamate. If copper ions are available, a bis(diethyldithiocarbamate)-copper(II) complex is formed. Disulfiram's selective anticancer activity is attributed to the copper(II) complex's ability to inhibit the cellular proteasome. It is assumed that the complex inhibits the proteasome by a mechanism that is distinct to the clinically used drug bortezomib, targeting the 19S rather than the 20S proteasome. This difference could be explained by inhibition of the JAMM domain of the POH1 subunit within the lid of the 19S proteasome.
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http://dx.doi.org/10.2174/138955712802762068DOI Listing
October 2012
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