Publications by authors named "Karel Soucek"

90 Publications

Phenotypic Heterogeneity of Triple-Negative Breast Cancer Mediated by Epithelial-Mesenchymal Plasticity.

Cancers (Basel) 2021 May 2;13(9). Epub 2021 May 2.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, 612 65 Brno, Czech Republic.

Triple-negative breast cancer (TNBC) is a subtype of breast carcinoma known for its unusually aggressive behavior and poor clinical outcome. Besides the lack of molecular targets for therapy and profound intratumoral heterogeneity, the relatively quick overt metastatic spread remains a major obstacle in effective clinical management. The metastatic colonization of distant sites by primary tumor cells is affected by the microenvironment, epigenetic state of particular subclones, and numerous other factors. One of the most prominent processes contributing to the intratumoral heterogeneity is an epithelial-mesenchymal transition (EMT), an evolutionarily conserved developmental program frequently hijacked by tumor cells, strengthening their motile and invasive features. In response to various intrinsic and extrinsic stimuli, malignant cells can revert the EMT state through the mesenchymal-epithelial transition (MET), a process that is believed to be critical for the establishment of macrometastasis at secondary sites. Notably, cancer cells rarely undergo complete EMT and rather exist in a continuum of E/M intermediate states, preserving high levels of plasticity, as demonstrated in primary tumors and, ultimately, in circulating tumor cells, representing a simplified element of the metastatic cascade. In this review, we focus on cellular drivers underlying EMT/MET phenotypic plasticity and its detrimental consequences in the context of TNBC cancer.
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http://dx.doi.org/10.3390/cancers13092188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125677PMC
May 2021

Skp2 and Slug Are Coexpressed in Aggressive Prostate Cancer and Inhibited by Neddylation Blockade.

Int J Mol Sci 2021 Mar 11;22(6). Epub 2021 Mar 11.

Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, 779 00 Olomouc, Czech Republic.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.
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http://dx.doi.org/10.3390/ijms22062844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000894PMC
March 2021

Highly selective inhibitors of protein kinases CLK and HIPK with the furo[3,2-b]pyridine core.

Eur J Med Chem 2021 Apr 18;215:113299. Epub 2021 Feb 18.

Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic; International Clinical Research Center, Center for Biomolecular and Cellular Engineering, St. Anne's University Hospital in Brno, Pekařská 53, 656 91, Brno, Czech Republic. Electronic address:

The furo [3,2-b]pyridine motif represents a relatively underexplored central pharmacophore in the area of kinase inhibitors. Herein, we report flexible synthesis of 3,5-disubstituted furo [3,2-b]pyridines that relies on chemoselective couplings of newly prepared 5-chloro-3-iodofuro [3,2-b]pyridine. This methodology allowed efficient second-generation synthesis of the state-of-the-art chemical biology probe for CLK1/2/4 MU1210, and identification of the highly selective inhibitors of HIPKs MU135 and MU1787 which are presented and characterized in this study, including the X-ray crystal structure of MU135 in HIPK2. chemical biology probe.
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http://dx.doi.org/10.1016/j.ejmech.2021.113299DOI Listing
April 2021

A prolonged exposure of human lung carcinoma epithelial cells to benzo[a]pyrene induces p21-dependent epithelial-to-mesenchymal transition (EMT)-like phenotype.

Chemosphere 2021 Jan 10;263:128126. Epub 2020 Sep 10.

Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic. Electronic address:

Deciphering the role of the aryl hydrocarbon receptor (AhR) in lung cancer cells may help us to better understand the role of toxic AhR ligands in lung carcinogenesis, including cancer progression. We employed human lung carcinoma A549 cells to investigate their fate after continuous two-week exposure to model AhR agonists, genotoxic benzo[a]pyrene (BaP; 1 μM) and non-genotoxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM). While TCDD increased proliferative rate of A549 cells, exposure to BaP decreased cell proliferation and induced epithelial-to-mesenchymal transition (EMT)-like phenotype, which was associated with enhanced cell migration, invasion, and altered cell morphology. Although TCDD also suppressed expression of E-cadherin and activated some genes linked to EMT, it did not induce the EMT-like phenotype. The results of transcriptomic analysis, and the opposite effects of BaP and TCDD on cell proliferation, indicated that a delay in cell cycle progression, together with a slight increase of senescence (when coupled with AhR activation), favors the induction of EMT-like phenotype. The shift towards EMT-like phenotype observed after simultaneous treatment with TCDD and mitomycin C (an inhibitor of cell proliferation) confirmed the hypothesis. Since BaP decreased cell proliferative rate via induction of p21 expression, we generated the A549 cell model with reduced p21 expression and exposed it to BaP for two weeks. The p21 knockdown suppressed the BaP-mediated EMT-like phenotype in A549 cells, thus confirming that a delayed cell cycle progression, together with p21-dependent induction of senescence-related chemokine CCL2, may contribute to induction of EMT-like cell phenotype in lung cells exposed to genotoxic AhR ligands.
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http://dx.doi.org/10.1016/j.chemosphere.2020.128126DOI Listing
January 2021

Trop2: Jack of All Trades, Master of None.

Cancers (Basel) 2020 Nov 11;12(11). Epub 2020 Nov 11.

Department of Experimental Biology, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic.

Trophoblast cell surface antigen 2 (Trop2) is a widely expressed glycoprotein and an epithelial cell adhesion molecule (EpCAM) family member. Although initially identified as a transmembrane protein, other subcellular localizations and processed forms were described. Its congenital mutations cause a gelatinous drop-like corneal dystrophy, a disease characterized by loss of barrier function in corneal epithelial cells. Trop2 is considered a stem cell marker and its expression associates with regenerative capacity in various tissues. Trop2 overexpression was described in tumors of different origins; however, functional studies revealed both oncogenic and tumor suppressor roles. Nevertheless, therapeutic potential of Trop2 was recognized and clinical studies with drug-antibody conjugates have been initiated in various cancer types. One of these agents, sacituzumab govitecan, has been recently granted an accelerated approval for therapy of metastatic triple-negative breast cancer. In this article, we review the current knowledge about the yet controversial function of Trop2 in homeostasis and pathology.
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http://dx.doi.org/10.3390/cancers12113328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696911PMC
November 2020

Toll-Like Receptor 3 in Solid Cancer and Therapy Resistance.

Cancers (Basel) 2020 Nov 2;12(11). Epub 2020 Nov 2.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, 612 65 Brno, Czech Republic.

Toll-like receptor 3 (TLR3) is a member of the TLR family, which has been extensively studied for its antiviral function. It is highly expressed in the endosomes of antigen-presenting immune cells and epithelial cells. TLR3 binds specifically double-strand RNAs (dsRNAs), leading to the activation of mainly two downstream pathways: the phosphorylation of IRF3, with subsequent production of type I interferon, and the activation of NF-κB, which drives the production of inflammatory cytokines and chemokines. Several studies have demonstrated TLR3 expression in multiple neoplasia types including breast, prostate, and lung cancer. Most studies were focused on the beneficial role of TLR3 activation in tumor cells, which leads to the production of cytotoxic cytokines and interferons and promotes caspase-dependent apoptosis. Indeed, ligands of this receptor were proposed for the treatment of cancer, also in combination with conventional chemotherapy. In contrast to these findings, recent evidence showed a link between TLR3 and tumor progression, metastasis, and therapy resistance. In the present review, we summarize the current knowledge of the mechanisms through which TLR3 can either lead to tumor regression or promote carcinogenesis as well as the potential of TLR-based therapies in resistant cancer.
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http://dx.doi.org/10.3390/cancers12113227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692054PMC
November 2020

3D Cell Culture Models Demonstrate a Role for FGF and WNT Signaling in Regulation of Lung Epithelial Cell Fate and Morphogenesis.

Front Cell Dev Biol 2020 21;8:574. Epub 2020 Jul 21.

Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czechia.

FGF signaling plays an essential role in lung development, homeostasis, and regeneration. We employed mouse 3D cell culture models and imaging to study the role of FGF ligands and the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways in lung epithelial morphogenesis and differentiation. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In a 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation. FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPC survival and/or initial divisions. FGF7 and FGF10 produced bigger organoids and induced organoid branching with higher frequency than FGF2 or FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but non-essential role in FGF-induced lung organoid formation. Analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. FGF ligands showed differences in promoting distinct lung epithelial cell types. FGF9 was a potent inducer of more proximal cell types, including ciliated and basal cells. FGF7 and FGF10 directed the differentiation toward distal lung lineages. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in postnatal lung development and homeostasis, and we reveal distinct roles for FGF ligands in regulation of mouse lung morphogenesis and differentiation .
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http://dx.doi.org/10.3389/fcell.2020.00574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396690PMC
July 2020

TGF-β regulates Sca-1 expression and plasticity of pre-neoplastic mammary epithelial stem cells.

Sci Rep 2020 07 9;10(1):11396. Epub 2020 Jul 9.

Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 612 65, Brno, Czech Republic.

The epithelial-mesenchymal plasticity, in tight association with stemness, contributes to the mammary gland homeostasis, evolution of early neoplastic lesions and cancer dissemination. Focused on cell surfaceome, we used mouse models of pre-neoplastic mammary epithelial and cancer stem cells to reveal the connection between cell surface markers and distinct cell phenotypes. We mechanistically dissected the TGF-β family-driven regulation of Sca-1, one of the most commonly used adult stem cell markers. We further provided evidence that TGF-β disrupts the lineage commitment and promotes the accumulation of tumor-initiating cells in pre-neoplastic cells.
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http://dx.doi.org/10.1038/s41598-020-67827-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347574PMC
July 2020

Slug-expressing mouse prostate epithelial cells have increased stem cell potential.

Stem Cell Res 2020 07 12;46:101844. Epub 2020 May 12.

Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 612 65 Brno, Czech Republic; Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital Brno, Pekařská 53, 656 91 Brno, Czech Republic; Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic. Electronic address:

Deciphering the properties of adult stem cells is crucial for understanding of their role in healthy tissue and in cancer progression as well. Both stem cells and cancer stem cells have shown association with epithelial-to-mesenchymal transition (EMT) in various tissue types. Aiming to investigate the epithelial and mesenchymal phenotypic traits in adult mouse prostate, we sorted subpopulations of basal prostate stem cells (mPSCs) and assessed the expression levels of EMT regulators and markers with custom-designed gene expression array. The population of mPSCs defined by a Lin/Sca-1CD49f/Trop-2 (LSC Trop-2) surface phenotype was enriched in mesenchymal markers, especially EMT master regulator Slug, encoded by the Snai2 gene. To further dissect the role of Slug in mPSCs, we used transgenic Snai2 reporter mouse strain. Using this model, we confirmed the presence of mesenchymal traits and increase of organoid forming capacity in Slug population of mPSCs. The Slug-derived organoids comprised all prostate epithelial cell types - basal, luminal, and neuroendocrine. Collectively, these data uncover the important role of Slug expression in the physiology of mouse prostate stem cells.
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http://dx.doi.org/10.1016/j.scr.2020.101844DOI Listing
July 2020

The CHK1 inhibitor MU380 significantly increases the sensitivity of human docetaxel-resistant prostate cancer cells to gemcitabine through the induction of mitotic catastrophe.

Mol Oncol 2020 10 16;14(10):2487-2503. Epub 2020 Jul 16.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality; hence, CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here, we report that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine (GEM) and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant (DR) mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to GEM in a panel of chemo-naïve and matched DR PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with GEM than clinical candidate SCH900776. MU380 alone or in combination with GEM significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in DR models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a prestage of apoptosis. Finally, treatment by MU380 alone, or in combination with GEM, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of GEM in DR mCRPC models. This approach might allow for dose reduction of GEM and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable DR mCRPC.
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http://dx.doi.org/10.1002/1878-0261.12756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530791PMC
October 2020

Ring-Substituted 1-Hydroxynaphthalene-2-Carboxanilides Inhibit Proliferation and Trigger Mitochondria-Mediated Apoptosis.

Int J Mol Sci 2020 May 12;21(10). Epub 2020 May 12.

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1, 612 42 Brno, Czech Republic.

Ring-substituted 1-hydroxynaphthalene-2-carboxanilides were previously investigated for their antimycobacterial properties. In our study, we have shown their antiproliferative and cell death-inducing effects in cancer cell lines. Cell proliferation and viability were assessed by WST-1 assay and a dye exclusion test, respectively. Cell cycle distribution, phosphatidylserine externalization, levels of reactive oxygen or nitrogen species (RONS), mitochondrial membrane depolarization, and release of cytochrome c were estimated by flow cytometry. Levels of regulatory proteins were determined by Western blotting. Our data suggest that the ability to inhibit the proliferation of THP-1 or MCF-7 cells might be referred to - or -substituted derivatives with electron-withdrawing groups -F, -Br, or -CF at anilide moiety. This effect was accompanied by accumulation of cells in G1 phase. Compound also induced apoptosis in THP-1 cells in association with a loss of mitochondrial membrane potential and production of mitochondrial superoxide. Our study provides a new insight into the action of salicylanilide derivatives, hydroxynaphthalene carboxamides, in cancer cells. Thus, their structure merits further investigation as a model moiety of new small-molecule compounds with potential anticancer properties.
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http://dx.doi.org/10.3390/ijms21103416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279329PMC
May 2020

ZEB1: A Critical Regulator of Cell Plasticity, DNA Damage Response, and Therapy Resistance.

Front Mol Biosci 2020 19;7:36. Epub 2020 Mar 19.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czechia.

The predominant way in which conventional chemotherapy kills rapidly proliferating cancer cells is the induction of DNA damage. However, chemoresistance remains the main obstacle to therapy effectivity. An increasing number of studies suggest that epithelial-to-mesenchymal transition (EMT) represents a critical process affecting the sensitivity of cancer cells to chemotherapy. Zinc finger E-box binding homeobox 1 (ZEB1) is a prime element of a network of transcription factors controlling EMT and has been identified as an important molecule in the regulation of DNA damage, cancer cell differentiation, and metastasis. Recent studies have considered upregulation of ZEB1 as a potential modulator of chemoresistance. It has been hypothesized that cancer cells undergoing EMT acquire unique properties that resemble those of cancer stem cells (CSCs). These stem-like cells manifest enhanced DNA damage response (DDR) and DNA repair capacity, self-renewal, or chemoresistance. In contrast, functional experiments have shown that ZEB1 induces chemoresistance regardless of whether other EMT-related changes occur. ZEB1 has also been identified as an important regulator of DDR by the formation of a ZEB1/p300/PCAF complex and direct interaction with ATM kinase, which has been linked to radioresistance. Moreover, ATM can directly phosphorylate ZEB1 and enhance its stability. Downregulation of ZEB1 has also been shown to reduce the abundance of CHK1, an effector kinase of DDR activated by ATR, and to induce its ubiquitin-dependent degradation. In this perspective, we focus on the role of ZEB1 in the regulation of DDR and describe the mechanisms of ZEB1-dependent chemoresistance.
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http://dx.doi.org/10.3389/fmolb.2020.00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096573PMC
March 2020

Blind deconvolution estimation of an arterial input function for small animal DCE-MRI.

Magn Reson Imaging 2019 10 28;62:46-56. Epub 2019 May 28.

Institute of Scientific Instruments of the Czech Academy of Sciences, Kralovopolska 147, 61264 Brno, Czech Republic.

Purpose: One of the main obstacles for reliable quantitative dynamic contrast-enhanced (DCE) MRI is the need for accurate knowledge of the arterial input function (AIF). This is a special challenge for preclinical small animal applications where it is very difficult to measure the AIF without partial volume and flow artifacts. Furthermore, using advanced pharmacokinetic models (allowing estimation of blood flow and permeability-surface area product in addition to the classical perfusion parameters) poses stricter requirements on the accuracy and precision of AIF estimation. This paper addresses small animal DCE-MRI with advanced pharmacokinetic models and presents a method for estimation of the AIF based on blind deconvolution.

Methods: A parametric AIF model designed for small animal physiology and use of advanced pharmacokinetic models is proposed. The parameters of the AIF are estimated using multichannel blind deconvolution.

Results: Evaluation on simulated data show that for realistic signal to noise ratios blind deconvolution AIF estimation leads to comparable results as the use of the true AIF. Evaluation on real data based on DCE-MRI with two contrast agents of different molecular weights showed a consistence with the known effects of the molecular weight.

Conclusion: Multi-channel blind deconvolution using the proposed AIF model specific for small animal DCE-MRI provides reliable perfusion parameter estimates under realistic signal to noise conditions.
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http://dx.doi.org/10.1016/j.mri.2019.05.024DOI Listing
October 2019

Novel CHK1 inhibitor MU380 exhibits significant single-agent activity in TP53-mutated chronic lymphocytic leukemia cells.

Haematologica 2019 12 11;104(12):2443-2455. Epub 2019 Apr 11.

Department of Internal Medicine, Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University

Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both -wild type and -mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both -mut), and OSU-CLL (-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction ( mutations or mutations) and -wt/-wt cells. Lastly, MU380 also exhibited significant activity in a xenotransplant mouse model (immunodeficient strain NOD- IL2R ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.
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http://dx.doi.org/10.3324/haematol.2018.203430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959166PMC
December 2019

High Skp2 expression is associated with a mesenchymal phenotype and increased tumorigenic potential of prostate cancer cells.

Sci Rep 2019 04 5;9(1):5695. Epub 2019 Apr 5.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

Skp2 is a crucial component of SCF E3 ubiquitin ligase and is often overexpressed in various types of cancer, including prostate cancer (PCa). The epithelial-to-mesenchymal transition (EMT) is involved in PCa progression. The acquisition of a mesenchymal phenotype that results in a cancer stem cell (CSC) phenotype in PCa was described. Therefore, we aimed to investigate the expression and localization of Skp2 in clinical samples from patients with PCa, the association of Skp2 with EMT status, and the role of Skp2 in prostate CSC. We found that nuclear expression of Skp2 was increased in patients with PCa compared to those with benign hyperplasia, and correlated with high Gleason score in PCa patients. Increased Skp2 expression was observed in PCa cell lines with mesenchymal and CSC-like phenotype compared to their epithelial counterparts. Conversely, the CSC-like phenotype was diminished in cells in which SKP2 expression was silenced. Furthermore, we observed that Skp2 downregulation led to the decrease in subpopulation of CD44CD24 cancer stem-like cells. Finally, we showed that high expression levels of both CD24 and CD44 were associated with favorable recurrence-free survival for PCa patients. This study uncovered the Skp2-mediated CSC-like phenotype with oncogenic functions in PCa.
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http://dx.doi.org/10.1038/s41598-019-42131-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451010PMC
April 2019

Generation of human iPSCs from fetal prostate fibroblasts HPrF.

Stem Cell Res 2019 03 7;35:101405. Epub 2019 Feb 7.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic. Electronic address:

Human induced pluripotent stem cell line was generated from commercially available primary human prostate fibroblasts HPrF derived from a fetus, aged 18-24 weeks of gestation. The fibroblast cell line was reprogrammed with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting the expression of factors of pluripotency on a single-cell level, and in vivo using teratoma formation assay. This iPS cell line will be a useful tool for studying both normal prostate development and prostate cancer disease.
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http://dx.doi.org/10.1016/j.scr.2019.101405DOI Listing
March 2019

LC-MS/MS study of in vivo fate of hyaluronan polymeric micelles carrying doxorubicin.

Carbohydr Polym 2019 Apr 3;209:181-189. Epub 2019 Jan 3.

Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic.

A better understanding of in vivo behavior of nanocarriers is necessary for further improvement in their development. Here we present a novel approach, where both the matrix and the drug can be analyzed by LCMS/MS after one sample handling. The developed method was applied for the comparison of pharmacokinetic profile of free and encapsulated doxorubicin (DOX) in oleyl hyaluronan (HA-C18:1) polymeric micelles. The results indicated that nanocarriers were rapidly dissociated upon in vivo administration. Despite this fact, the administration of encapsulated DOX led to its longer circulation time and enhanced tumor targeting. This effect was not observed injecting blank HA-C18:1 micelles followed by unencapsulated DOX. Biodistribution studies and molecular weight estimation of the carrier matrix indicated relatively high stability of HA-C18:1 ester bond in bloodstream and complete elimination of the derivative within 72 h. The proposed methodology provides a novel strategy to elucidate the pharmacokinetic behavior of polysaccharide-based drug delivery systems.
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http://dx.doi.org/10.1016/j.carbpol.2018.12.104DOI Listing
April 2019

Furo[3,2-b]pyridine: A Privileged Scaffold for Highly Selective Kinase Inhibitors and Effective Modulators of the Hedgehog Pathway.

Angew Chem Int Ed Engl 2019 01 20;58(4):1062-1066. Epub 2018 Dec 20.

Department of Chemistry, CZ-Openscreen, Masaryk University, Kamenice 5, Brno, 625 00, Czech Republic.

Reported is the identification of the furo[3,2-b]pyridine core as a novel scaffold for potent and highly selective inhibitors of cdc-like kinases (CLKs) and efficient modulators of the Hedgehog signaling pathway. Initially, a diverse target compound set was prepared by synthetic sequences based on chemoselective metal-mediated couplings, including assembly of the furo[3,2-b]pyridine scaffold by copper-mediated oxidative cyclization. Optimization of the subseries containing 3,5-disubstituted furo[3,2-b]pyridines afforded potent, cell-active, and highly selective inhibitors of CLKs. Profiling of the kinase-inactive subset of 3,5,7-trisubstituted furo[3,2-b]pyridines revealed sub-micromolar modulators of the Hedgehog pathway.
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http://dx.doi.org/10.1002/anie.201810312DOI Listing
January 2019

Generation of human iPSCs from human prostate cancer-associated fibroblasts IBPi002-A.

Stem Cell Res 2018 12 16;33:255-259. Epub 2018 Nov 16.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic. Electronic address:

A human induced pluripotent stem cell line was generated from cancer-associated fibroblasts of a 68-years old patient with diagnosed prostate adenocarcinoma (PCa). The fibroblast cell line was reprogrammed with Epi5™ Episomal iPSC Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting expression of factors of pluripotency on a single-cell level, and also in vivo using teratoma formation assay. This new iPS cell line may be used for differentiation into different prostate-specific cell types in differentiation studies.
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http://dx.doi.org/10.1016/j.scr.2018.11.006DOI Listing
December 2018

In vivo monitoring of tumor distribution of hyaluronan polymeric micelles labeled or loaded with near-infrared fluorescence dye.

Carbohydr Polym 2018 Oct 22;198:339-347. Epub 2018 Jun 22.

Contipro a.s., Dolní Dobrouč 401, Dolní Dobrouč, 561 02, Czech Republic.

Development of delivery systems which allow real-time visual inspection of tumors is critical for effective therapy. Near-infrared (NIR) fluorophores have a great potential for such an application. To overcome NIR dyes short blood circulation time and increase tumor accumulation, a NIR dye, cypate, was associated with oleyl hyaluronan, which can self-assemble into polymeric aggregates. The cypate association with oleyl hyaluronan was performed either by a covalent linkage, or physical entrapment. The two systems were compared for tumor targeting and contrast enhancement using BALB/c mice bearing 4T1 breast cancer tumors. Independently on the way of cypate association, it took more than 24 h from intravenous administration to detect NIR signal in tumors and the tumors were clearly visualized for 2 following weeks without substrate reinjection. Covalently linked cypate generated 2-3 fold stronger fluorescence signal than physically loaded cypate. This study demonstrates the potential of HA matrix to be used as carrier of contrast agents for non-invasive long-term tumor visualization.
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http://dx.doi.org/10.1016/j.carbpol.2018.06.082DOI Listing
October 2018

Correction Alternative mechanisms of miR-34a regulation in cancer.

Cell Death Dis 2018 Jul 16;9(8):783. Epub 2018 Jul 16.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

The PDF and HTML versions of the article have been updated to include the Creative Commons Attribution 4.0 International License information.
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http://dx.doi.org/10.1038/s41419-018-0833-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048027PMC
July 2018

Trop-2 plasticity is controlled by epithelial-to-mesenchymal transition.

Carcinogenesis 2018 12;39(11):1411-1418

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

The cell surface glycoprotein Trop-2 is commonly overexpressed in carcinomas and represents an exceptional antigen for targeted therapy. Here, we provide evidence that surface Trop-2 expression is functionally connected with an epithelial phenotype in breast and prostate cell lines and in patient tumor samples. We further show that Trop-2 expression is suppressed epigenetically or through the action of epithelial-to-mesenchymal transition transcription factors and that deregulation of Trop-2 expression is linked with cancer progression and poor patient prognosis. Moreover, our data suggest that the cancer plasticity-driven intratumoral heterogeneity in Trop-2 expression may significantly contribute to response and resistance to therapies targeting Trop-2-expressing cells.
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http://dx.doi.org/10.1093/carcin/bgy095DOI Listing
December 2018

Presence of growth/differentiation factor-15 cytokine in human follicular fluid, granulosa cells, and oocytes.

J Assist Reprod Genet 2018 Aug 13;35(8):1407-1417. Epub 2018 Jun 13.

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

Purpose: The purpose of the study was to determine whether the GDF-15 is present in follicular fluid; to evaluate if there is a relation between follicular and serum levels of GDF-15 and fertility status of study subjects; and to test whether granulosa cells, oocytes, or both produce GDF-15.

Methods: This study used follicular fluid (FF, serum, and oocytes obtained under informed consent from women undergoing oocyte retrieval for in vitro fertilization. It also used ovaries from deceased preterm newborns. Collection of FF and blood at the time of oocyte retrieval, ELISA and western blot were performed to determine levels and forms of GDF-15. Concentrations of GDF-15 in FF and serum, its expression in ovarian tissue, and secretion from granulosa cells were analyzed.

Results: GDF-15 concentration in FF ranged from 35 to 572 ng/ml, as determined by ELISA. Western blot analysis revealed the GDF-15 pro-dimer only in FF. Both normal healthy and cancerous granulosa cells secreted GDF-15 into culture media. Primary oocytes displayed cytoplasmic GDF-15 positivity in immunostained newborn ovaries, and its expression was also observed in fully grown human oocytes.

Conclusions: To the best of our knowledge, this is the first documentation of cytokine GDF-15 presence in follicular fluid. Its concentration was not associated with donor/patient fertility status. Our data also show that GDF-15 is expressed and inducible in both normal healthy and cancerous granulosa cells, as well as in oocytes.
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http://dx.doi.org/10.1007/s10815-018-1230-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086784PMC
August 2018

Soluble Cripto-1 Induces Accumulation of Supernumerary Centrosomes and Formation of Aberrant Mitoses in Human Embryonic Stem Cells.

Stem Cells Dev 2018 08 17;27(16):1077-1084. Epub 2018 Jul 17.

1 Department of Histology and Embryology, Faculty of Medicine, Masaryk University , Brno, Czech Republic .

Chromosomal instability evoked by abnormalities in centrosome numbers has been traditionally considered as a hallmark of aberrant, typically cancerous or senescent cells. We have reported previously that pristine human embryonic stem cells (hESC) suffer from high frequency of supernumerary centrosomes and hence may be prone to undergo abnormal mitotic divisions. We have also unraveled that this phenomenon of multicentrosomal mitoses vanishes with prolonged time in culture and with initiation of differentiation, and it is strongly affected by the culture substratum. In this study, we report for the first time that Cripto-1 protein (teratocarcinoma-derived growth factor 1, epidermal growth factor-Cripto/FRL-1/Cryptic) produced by hESC represents a factor capable of inducing formation of supernumerary centrosomes in cultured hESC. Elimination of Cripto-1 signaling on the other hand restores the normal number of centrosomes in hESC. Linking the secretory phenotype of hESC to the centrosomal metabolism may help to develop better strategies for propagation of stable and safe bioindustrial and clinical grade cultures of hESC. From a broader point of view, it may lead to unravelling Cripto-1 as a micro-environmental factor contributing to adverse cell behaviors in vivo.
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http://dx.doi.org/10.1089/scd.2018.0017DOI Listing
August 2018

Hypericin affects cancer side populations via competitive inhibition of BCRP.

Biomed Pharmacother 2018 Mar 20;99:511-522. Epub 2018 Feb 20.

Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Šrobárova 2, 041 54, Košice, Slovak Republic. Electronic address:

Objective: Cancer stem-like cells (CSLCs) are considered a root of tumorigenicity and resistance. However, their identification remains challenging. The use of the side population (SP) assay as a credible marker of CSLCs remains controversial. The SP assay relies on the elevated activity of ABC transporters that, in turn, can be modulated by hypericin (HYP), a photosensitizer and bioactive compound of St. John's Wort (Hypericum perforatum), a popular over-the-counter antidepressant. Here we aimed to comprehensively characterize the SP phenotype of cancer cells and to determine the impact of HYP on these cells.

Methods: Flow cytometry and sorting-based assays were employed, including CD24-, CD44-, CD133-, and ALDH-positivity, clonogenicity, 3D-forming ability, ABC transporter expression and activity, and intracellular accumulation of HYP/Hoechst 33342. The tumorigenic ability of SP, nonSP, and HYP-treated cells was verified by xenotransplantation into immunodeficient mice.

Results: The SP phenotype was associated with elevated expression of several investigated transporters and more intensive growth in non-adherent conditions but not with higher clonogenicity, tumorigenicity or ALDH-positivity. Despite stimulated BCRP level and MRP1 activity, HYP reversibly decreased the SP proportion, presumably via competitive inhibition of BCRP. HYP-selected SP cells acquired additional traits of resistance and extensively eliminated HYP.

Conclusions: Our results suggest that SP is not an unequivocal CSLC-marker. However, SP could play an important role in modulating HYP-treatment and serve as a negative predictive tool for HYP-based therapies. Moreover, the use of supplements containing HYP by cancer patients should be carefully considered, due to its proposed effect on drug efflux and complex impact on tumor cells, which have not yet been sufficiently characterized.
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http://dx.doi.org/10.1016/j.biopha.2018.01.074DOI Listing
March 2018

Plasticity and intratumoural heterogeneity of cell surface antigen expression in breast cancer.

Br J Cancer 2018 03 20;118(6):813-819. Epub 2018 Feb 20.

Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, Brno 612 65, Czech Republic.

Background:The intratumoural heterogeneity, often driven by epithelial-to-mesenchymal transition (EMT), significantly contributes to chemoresistance and disease progression in adenocarcinomas. Methods:We introduced a high-throughput screening platform to identify surface antigens that associate with epithelial–mesenchymal plasticity in well-defined pairs of epithelial cell lines and their mesenchymal counterparts. Using multicolour flow cytometry, we then analysed the expression of 10 most robustly changed antigens and identified a 10-molecule surface signature, in pan-cytokeratin-positive/EpCAM-positive and -negative fractions of dissociated breast tumours. Results:We found that surface CD9, CD29, CD49c, and integrin ß5 are lost in breast cancer cells that underwent EMT in vivo. The tetraspanin family member CD9 was concordantly downregulated both in vitro and in vivo and associated with epithelial phenotype and favourable prognosis. Conclusions:We propose that overall landscape of 10-molecule surface signature expression reflects the epithelial–mesenchymal plasticity in breast cancer.
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http://dx.doi.org/10.1038/bjc.2017.497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886127PMC
March 2018

Metabolic stress regulates ERK activity by controlling KSR-RAF heterodimerization.

EMBO Rep 2018 02 20;19(2):320-336. Epub 2017 Dec 20.

International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the or oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS-mutant cells, and with oncogenic BRAF in BRAF-mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS-mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF-mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.
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http://dx.doi.org/10.15252/embr.201744524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797961PMC
February 2018

Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells.

PLoS One 2017 11;12(12):e0188772. Epub 2017 Dec 11.

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188772PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724894PMC
January 2018

Multiparameter cytometric analysis of complex cellular response.

Cytometry A 2018 02 8;93(2):239-248. Epub 2017 Dec 8.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

Complex analysis of cellular responses after experimental treatment is important for screening, mechanistic understanding of treatment effects, and the identification of sensitive and resistant cell phenotypes. Modern multicolor flow cytometry has demonstrated its power for such analyses. Here, we introduce a multiparametric protocol for complex analysis of cytokinetics by the simultaneous detection of seven fluorescence parameters. This analysis includes the detection of two surface markers for immunophenotyping, analysis of proliferation based on the cell cycle and the measurement of incorporated nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) in newly synthesized DNA, analysis of DNA damage using an anti-phospho-histone H2A.X (Ser139) antibody, and determination of cell death using a fixable viability probe and intracellular detection of caspase-3 activation. To demonstrate the applicability of this protocol for the analysis of heterogeneous and complex cell responses, we used different treatments and model cell lines. We demonstrated that this protocol has the potential to provide complex and simultaneous analysis of cytokinetics and analyze the heterogeneity of the response at the single-cell level. © 2017 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23295DOI Listing
February 2018

BRCA1 or CDK12 loss sensitizes cells to CHK1 inhibitors.

Tumour Biol 2017 Oct;39(10):1010428317727479

1 Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic.

A broad spectrum of tumors develop resistance to classic chemotherapy, necessitating the discovery of new therapies. One successful strategy exploits the synthetic lethality between poly(ADP-ribose) polymerase 1/2 proteins and DNA damage response genes, including BRCA1, a factor involved in homologous recombination-mediated DNA repair, and CDK12, a transcriptional kinase known to regulate the expression of DDR genes. CHK1 inhibitors have been shown to enhance the anti-cancer effect of DNA-damaging compounds. Since loss of BRCA1 increases replication stress and leads to DNA damage, we tested a hypothesis that CDK12- or BRCA1-depleted cells rely extensively on S-phase-related CHK1 functions for survival. The silencing of BRCA1 or CDK12 sensitized tumor cells to CHK1 inhibitors in vitro and in vivo. BRCA1 downregulation combined with CHK1 inhibition induced excessive amounts of DNA damage, resulting in an inability to complete the S-phase. Therefore, we suggest CHK1 inhibition as a strategy for targeting BRCA1- or CDK12-deficient tumors.
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http://dx.doi.org/10.1177/1010428317727479DOI Listing
October 2017