António R. Pombinho

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto

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Petr Bartůněk

Czech Academy of Sciences, Institute of Molecular Genetics

André F. Maia

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto

Olga Machoňová

Czech Academy of Sciences, Institute of Molecular Genetics

Dietmar Gradl

Karlsruhe Institute of Technology

Monika Horázná

Czech Academy of Sciences, Institute of Molecular Genetics

Martina Vojtěchová

Czech Academy of Sciences, Institute of Molecular Genetics

Vı́tězslav Křı́ž

Czech Academy of Sciences, Institute of Molecular Genetics

Zbyněk Zdráhal

Central European Institute of Technology

Michaela Krausová

Czech Academy of Sciences

EŠ

Eva Šloncová

Czech Academy of Sciences

Cooperative Institutions

Universidade do Porto

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto

Charles University

Czech Academy of Sciences

Czech Academy of Sciences, Institute of Molecular Genetics

IPO Porto

Central European Institute of Technology

Instituto de Biologia Molecular e Celular

Institute of Molecular Genetics

University of Algarve

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Monensin as potential drug for treatment of SLeX-positive tumors

medRxiv (Cold Spring Harbor Laboratory) (2024)

Ana Filipa Costa Emanuel Senra Diana Campos Isabel Faria‐Ramos Liliana Santos-Ferreira

Abstract Colorectal (CRC) and gastric (GC) cancers remain the top lethal cancers and targeted therapies in this setting are still very limited. Sialyl Lewis X (SLeX), a cancer-associated glycan highly expressed in both CRC and GC, plays a crucial role in cancer cell dissemination and metastasis. Thus, presenting a promising but still underexplored therapeutic target. In this work, we performed a high-throughput screening (HTS) approach to identify potential inhibitors of SLeX expression on cancer cells. Two libraries including a total of 7836 compounds were screened and monensin emerged as a promising SLeX inhibitor. Monensin promoted structural alterations in the secretory pathway, particularly at the Golgi apparatus, impacting protein O -glycosylation and secretion. RNAseq transcriptomic analysis uncovered significant alterations in Gene Ontology (GO) terms associated with protein misfolding, target to the membrane, as well as, epithelial cell-cell adhesion protein. In vitro studies showed that, upon treatment with monensin, SLeX-positive cancer cells showed reduced viability, concomitant with decreased motility and invasive capacities. Using in vivo xenograft models of chick embryo chorioallantoic membrane (CAM) and nude mice, revealed that monensin reduced tumor formation and invasion. Pre-clinical validation using gastric cancer patient-derived organoids (PDOs) and organoid xenotransplants in mice further underscored the clinical potential of monensin in suppressing the growth of SLeX- positive tumors. Overall, our findings set the ground for further evaluation of monensin as a novel therapeutic agent in GC and CRC in the clinical setting.

Monensin

10.1101/2024.03.11.24304048

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Citations (1)

Supplementary Materials and Methods, Figures 1 - 9, Tables 1 - 5 from Monensin Inhibits Canonical Wnt Signaling in Human Colorectal Cancer Cells and Suppresses Tumor Growth in Multiple Intestinal Neoplasia Mice

Lucie Tůmová António R. Pombinho Martina Vojtěchová Jitka Stančíková Dietmar Gradl

PDF file - 515KB, Supplementary Materials and Methods. Supplementary Figure S1. Densitometric analysis of western blots shown in Figs. 2D (A), 3C (B) and 4D (C). Supplementary Figure S2. Monensin suppresses Wnt signaling activated by TALEN-induced disruption of the APC gene. Supplementary Figure S3. Monensin does not interfere with Dvl phoshorylation. Supplementary Figure S4. The inhibitory effect of monensin on Wnt signaling is not restricted to the vesicular acidity blockade. Supplementary Figure S5. Monensin inhibits aberrant Wnt signaling in CRC cells COLO320 and LS174T. Supplementary Figure S6. ROS concentration is not increased by monensin. Supplementary Figure S7. Membranous localization of beta-catenin in HCT116 cells. Supplementary Figure S8. Monensin does not inhibit beta-catenin acetylation. Supplementary Figure S9. Monensin does not change the fraction of proliferating cells in intestinal tumors. Supplementary Table S1. Sequences of primers used for qRT-PCR analyses. Supplementary Table S2. Monensin does not inhibit enzymatic activity of any of the tested protein kinases. Supplementary Table S3. qRT-PCR analysis of the TCF/beta-catenin target gene expression in CRC cells. Supplementary Table S4. Cell cycle analysis of SW480 and HCT116 cells. Supplementary Table S5. Analysis of phosphorylated amino acid residues in beta-catenin.

Monensin

10.1158/1535-7163.22499776

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Data from Monensin Inhibits Canonical Wnt Signaling in Human Colorectal Cancer Cells and Suppresses Tumor Growth in Multiple Intestinal Neoplasia Mice

Lucie Tůmová António R. Pombinho Martina Vojtěchová Jitka Stančíková Dietmar Gradl

<div>AbstractThe Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/β-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with β-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or β-catenin–induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of β-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling. Mol Cancer Ther; 13(4); 812–22. ©2014 AACR.</div>

10.1158/1535-7163.c.6536254.v1

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Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution

Scientific Reports (2015)

Michaela Liegertová Jiří Pergner Iryna Kozmiková Péter Fábián António R. Pombinho

Abstract Animals sense light primarily by an opsin-based photopigment present in a photoreceptor cell. Cnidaria are arguably the most basal phylum containing a well-developed visual system. The evolutionary history of opsins in the animal kingdom has not yet been resolved. Here, we study the evolution of animal opsins by genome-wide analysis of the cubozoan jellyfish Tripedalia cystophora , a cnidarian possessing complex lens-containing eyes and minor photoreceptors. A large number of opsin genes with distinct tissue- and stage-specific expression were identified. Our phylogenetic analysis unequivocally classifies cubozoan opsins as a sister group to c-opsins and documents lineage-specific expansion of the opsin gene repertoire in the cubozoan genome. Functional analyses provided evidence for the use of the Gs-cAMP signaling pathway in a small set of cubozoan opsins, indicating the possibility that the majority of other cubozoan opsins signal via distinct pathways. Additionally, these tests uncovered subtle differences among individual opsins, suggesting possible fine-tuning for specific photoreceptor tasks. Based on phylogenetic, expression and biochemical analysis we propose that rapid lineage- and species-specific duplications of the intron-less opsin genes and their subsequent functional diversification promoted evolution of a large repertoire of both visual and extraocular photoreceptors in cubozoans.

Opsin

Lineage (genetic)

10.1038/srep11885

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Mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells

Molecular Biology of the Cell (2023)

Marco Novais-Cruz António R. Pombinho Mafalda Sousa André F. Maia Hélder Maiato

Extensive DNA damage during mitosis promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells. A previously overlooked mechanism by which DNA damage during mitosis contributes to chromosomal instability and micronuclei formation is unveiled.

10.1091/mbc.e22-11-0518

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Benzimidazoles Downregulate Mdm2 and MdmX and Activate p53 in MdmX Overexpressing Tumor Cells

Molecules (2019)

Zuzana Mrkvová Stjepan Uldrijan António R. Pombinho Petr Bartůněk Iva Slaninová

Tumor suppressor p53 is mutated in about 50% of cancers. Most malignant melanomas carry wild-type p53, but p53 activity is often inhibited due to overexpression of its negative regulators Mdm2 or MdmX. We performed high throughput screening of 2448 compounds on A375 cells carrying p53 activity luciferase reporter construct to reveal compounds that promote p53 activity in melanoma. Albendazole and fenbendazole, two approved and commonly used benzimidazole anthelmintics, stimulated p53 activity and were selected for further studies. The protein levels of p53 and p21 increased upon the treatment with albendazole and fenbendazole, indicating activation of the p53–p21 pathway, while the levels of Mdm2 and MdmX decreased in melanoma and breast cancer cells overexpressing these proteins. We also observed a reduction of cell viability and changes of cellular morphology corresponding to mitotic catastrophe, i.e., G2/M cell cycle arrest of large multinucleated cells with disrupted microtubules. In summary, we established a new tool for testing the impact of small molecule compounds on the activity of p53 and used it to identify the action of benzimidazoles in melanoma cells. The drugs promoted the stability and transcriptional activity of wild-type p53 via downregulation of its negative regulators Mdm2 and MdmX in cells overexpressing these proteins. The results indicate the potential for repurposing the benzimidazole anthelmintics for the treatment of cancers overexpressing p53 negative regulators.

MDMX

10.3390/molecules24112152

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Citations (26)

Retinoic acid is a negative regulator of matrix Gla protein gene expression in teleost fish Sparus aurata

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms (2007)

Natércia Conceição Vincent Laizé B. Simões António R. Pombinho M. Leonor Cancela

Transcription

10.1016/j.bbagrm.2007.11.003

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Citations (19)

Colony stimulating factor 1 signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Martina Hason Tereza Mikulášová Olga Machoňová António R. Pombinho Tjakko J. van Ham

Abstract Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. Therapeutic targeting of the CSF1R pathway is a promising strategy in many human diseases, including neurological disorders or cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands - Csf1a, Csf1b and Il34, in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors as it is diminished in both csf1ra Δ5bp and csf1rb Δ 4bp mutants. There is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Further, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rb Δ4bp and il34 Δ5bp deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors and that the expression of csf1ra and csf1rb is non-overlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis. Key points: csf1ra and csf1rb are indispensable for macrophage differentiation and together with csf1a regulate embryonic macrophage fates in zebrafish csf1rb is important for granulocyte differentiation and migration and together with il34 it regulates embryonic granulocytic fates in zebrafish

Myelopoiesis

10.1101/2021.04.06.438628

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Correction: Corrigendum: Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution

Scientific Reports (2015)

Michaela Liegertová Jiří Pergner Iryna Kozmiková Péter Fábián António R. Pombinho

Animals sense light primarily by an opsin-based photopigment present in a photoreceptor cell. Cnidaria are arguably the most basal phylum containing a well-developed visual system. The evolutionary history of opsins in the animal kingdom has not yet been resolved. Here, we study the evolution of animal opsins by genome-wide analysis of the cubozoan jellyfish Tripedalia cystophora , a cnidarian possessing complex lens-containing eyes and minor photoreceptors. A large number of opsin genes with distinct tissue- and stage-specific expression were identified. Our phylogenetic analysis unequivocally classifies cubozoan opsins as a sister group to c-opsins and documents lineage-specific expansion of the opsin gene repertoire in the cubozoan genome. Functional analyses provided evidence for the use of the Gs-cAMP signaling pathway in a small set of cubozoan opsins, indicating the possibility that the majority of other cubozoan opsins signal via distinct pathways. Additionally, these tests uncovered subtle differences among individual opsins, suggesting possible fine-tuning for specific photoreceptor tasks. Based on phylogenetic, expression and biochemical analysis we propose that rapid lineage- and species-specific duplications of the intron-less opsin genes and their subsequent functional diversification promoted evolution of a large repertoire of both visual and extraocular photoreceptors in cubozoans.

Opsin

Lineage (genetic)

10.1038/srep14396

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Citations (2)

HMGA1 stimulates cancer stem-like features and sensitivity to monensin in gastric cancer

Experimental Cell Research (2024)

Diana Pádua Paula Figueira António R. Pombinho Inês Monteiro Carlos‐Filipe Pereira

Monensin

10.1016/j.yexcr.2024.114257

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