Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models.  CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

The Golgi checkpoint: Golgi unlinking during G2 is necessary for spindle formation and cytokinesis.

Entry into mitosis requires not only correct DNA replication but also extensive cell reorganization, including the separation of the Golgi ribbon into isolated stacks. To understand the significance of pre-mitotic Golgi reorganization, we devised a strategy to first block Golgi segregation, with the consequent G2-arrest, and then force entry into mitosis. We found that the cells forced to enter mitosis with an intact Golgi ribbon showed remarkable cell division defects, including spindle multipolarity and binucleation. The spindle defects were caused by reduced levels at the centrosome of the kinase Aurora-A, a pivotal spindle formation regulator controlled by Golgi segregation. Overexpression of Aurora-A rescued spindle formation, indicating a crucial role of the Golgi-dependent recruitment of Aurora-A at the centrosome. Thus, our results reveal that alterations of the pre-mitotic Golgi segregation in G2 have profound consequences on the fidelity of later mitotic processes and represent potential risk factors for cell transformation and cancer development.

In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon During the G2-M Transition of the Cell Cycle.

The Golgi complex is the central hub of the secretory pathway. In mammalian cells, it is formed by stacks of flattened cisternae organized in a continuous membrane system, the Golgi ribbon, located near the centrosome. During G2, the Golgi ribbon is disassembled into isolated stacks that, at the onset of mitosis, are further fragmented into small tubular-vesicular clusters that disperse throughout the cytoplasm. Here, we describe a set of methods to study the Golgi complex in different phases of the cell cycle, drawing attention to reproducing the mitotic Golgi fragmentation to gain knowledge and acquire the skills to study the mechanisms that regulate mitotic Golgi reorganization as well as its biological significance. The investigations based on these assays have been instrumental in understanding that Golgi disassembly is not only a consequence of mitosis but is also required for mitotic entry and cell division.

PARP10 Mediates Mono-ADP-Ribosylation of Aurora-A Regulating G2/M Transition of the Cell Cycle.

Intracellular mono-ADP-ribosyltransferases (mono-ARTs) catalyze the covalent attachment of a single ADP-ribose molecule to protein substrates, thus regulating their functions. PARP10 is a soluble mono-ART involved in the modulation of intracellular signaling, metabolism and apoptosis. PARP10 also participates in the regulation of the G1- and S-phase of the cell cycle. However, the role of this enzyme in G2/M progression is not defined. In this study, we found that genetic ablation, protein depletion and pharmacological inhibition of PARP10 cause a delay in the G2/M transition of the cell cycle. Moreover, we found that the mitotic kinase Aurora-A, a previously identified PARP10 substrate, is actively mono-ADP-ribosylated (MARylated) during G2/M transition in a PARP10-dependent manner. Notably, we showed that PARP10-mediated MARylation of Aurora-A enhances the activity of the kinase in vitro. Consistent with an impairment in the endogenous activity of Aurora-A, cells lacking PARP10 show a decreased localization of the kinase on the centrosomes and mitotic spindle during G2/M progression. Taken together, our data provide the first evidence of a direct role played by PARP10 in the progression of G2 and mitosis, an event that is strictly correlated to the endogenous MARylation of Aurora-A, thus proposing a novel mechanism for the modulation of Aurora-A kinase activity.

Structural Organization and Function of the Golgi Ribbon During Cell Division.

The Golgi complex has a central role in the secretory traffic. In vertebrate cells it is generally organized in polarized stacks of cisternae that are laterally connected by membranous tubules, forming a structure known as Golgi ribbon. The steady state ribbon arrangement results from a dynamic equilibrium between formation and cleavage of the membrane tubules connecting the stacks. This balance is of great physiological relevance as the unlinking of the ribbon during G2 is required for mitotic entry. A block of this process induces a potent G2 arrest of the cell cycle, indicating that a mitotic "Golgi checkpoint" controls the correct pre-mitotic segregation of the Golgi ribbon. Then, after mitosis onset, the Golgi stacks undergo an extensive disassembly, which is necessary for proper spindle formation. Notably, several Golgi-associated proteins acquire new roles in spindle formation and mitotic progression during mitosis. Here we summarize the current knowledge about the basic principle of the Golgi architecture and its functional relationship with cell division to highlight crucial aspects that need to be addressed to help us understand the physiological significance of the ribbon and the pathological implications of alterations of this organization.

Golgi Complex: A Signaling Hub in Cancer.

The Golgi Complex is the central hub in the endomembrane system and serves not only as a biosynthetic and processing center but also as a trafficking and sorting station for glycoproteins and lipids. In addition, it is an active signaling hub involved in the regulation of multiple cellular processes, including cell polarity, motility, growth, autophagy, apoptosis, inflammation, DNA repair and stress responses. As such, the dysregulation of the Golgi Complex-centered signaling cascades contributes to the onset of several pathological conditions, including cancer. This review summarizes the current knowledge on the signaling pathways regulated by the Golgi Complex and implicated in promoting cancer hallmarks and tumor progression.

Functional Coordination among the Golgi Complex, the Centrosome and the Microtubule Cytoskeleton during the Cell Cycle.

The Golgi complex of mammalian cells is organized in a ribbon-like structure often closely associated with the centrosome during interphase. Conversely, the Golgi complex assumes a fragmented and dispersed configuration away from the centrosome during mitosis. The structure of the Golgi complex and the relative position to the centrosome are dynamically regulated by microtubules. Many pieces of evidence reveal that this microtubule-mediated dynamic association between the Golgi complex and centrosome is of functional significance in cell polarization and division. Here, we summarize findings indicating how the Golgi complex and the centrosome cooperate in organizing the microtubule network for the directional protein transport and centrosome positioning required for cell polarization and regulating fundamental cell division processes.

Corrigendum: Combinatorial Strategies to Target Molecularand Signaling Pathways to Disarm Cancer Stem Cells.

[This corrects the article DOI: 10.3389/fonc.2021.689131.].

Combinatorial Strategies to Target Molecular and Signaling Pathways to Disarm Cancer Stem Cells.

Cancer is an urgent public health issue with a very huge number of cases all over the world expected to increase by 2040. Despite improved diagnosis and therapeutic protocols, it remains the main leading cause of death in the world. Cancer stem cells (CSCs) constitute a tumor subpopulation defined by ability to self-renewal and to generate the heterogeneous and differentiated cell lineages that form the tumor bulk. These cells represent a major concern in cancer treatment due to resistance to conventional protocols of radiotherapy, chemotherapy and molecular targeted therapy. In fact, although partial or complete tumor regression can be achieved in patients, these responses are often followed by cancer relapse due to the expansion of CSCs population. The aberrant activation of developmental and oncogenic signaling pathways plays a relevant role in promoting CSCs therapy resistance. Although several targeted approaches relying on monotherapy have been developed to affect these pathways, they have shown limited efficacy. Therefore, an urgent need to design alternative combinatorial strategies to replace conventional regimens exists. This review summarizes the preclinical studies which provide a proof of concept of therapeutic efficacy of combinatorial approaches targeting the CSCs.

The Golgi ribbon: mechanisms of maintenance and disassembly during the cell cycle.

The Golgi complex (GC) has an essential role in the processing and sorting of proteins and lipids. The GC of mammalian cells is composed of stacks of cisternae connected by membranous tubules to create a continuous network, the Golgi ribbon, whose maintenance requires several core and accessory proteins. Despite this complex structural organization, the Golgi apparatus is highly dynamic, and this property becomes particularly evident during mitosis, when the ribbon undergoes a multistep disassembly process that allows its correct partitioning and inheritance by the daughter cells. Importantly, alterations of the Golgi structure are associated with a variety of physiological and pathological conditions. Here, we review the core mechanisms and signaling pathways involved in both the maintenance and disassembly of the Golgi ribbon, and we also report on the signaling pathways that connect the disassembly of the Golgi ribbon to mitotic entry and progression.

Organelle Inheritance Control of Mitotic Entry and Progression: Implications for Tissue Homeostasis and Disease.

The Golgi complex (GC), in addition to its well-known role in membrane traffic, is also actively involved in the regulation of mitotic entry and progression. In particular, during the G2 phase of the cell cycle, the Golgi ribbon is unlinked into isolated stacks. Importantly, this ribbon cleavage is required for G2/M transition, indicating that a "Golgi mitotic checkpoint" controls the correct segregation of this organelle. Then, during mitosis, the isolated Golgi stacks are disassembled, and this process is required for spindle formation. Moreover, recent evidence indicates that also proper mitotic segregation of other organelles, such as mitochondria, endosomes, and peroxisomes, is required for correct mitotic progression and/or spindle formation. Collectively, these observations imply that in addition to the control of chromosomes segregation, which is required to preserve the genetic information, the cells actively monitor the disassembly and redistribution of subcellular organelles in mitosis. Here, we provide an overview of the major structural reorganization of the GC and other organelles during G2/M transition and of their regulatory mechanisms, focusing on novel findings that have shed light on the basic processes that link organelle inheritance to mitotic progression and spindle formation, and discussing their implications for tissue homeostasis and diseases.

GRASP65 controls Golgi position and structure during G2/M transition by regulating the stability of microtubules.

The mammalian Golgi apparatus is organized in the form of a ribbon-like structure positioned near the centrosome. Despite its multimodular organization, the Golgi complex is characterized by a prominent structural plasticity, which is crucial during essential physiological processes, such as the G2 phase of the cell cycle, during which the Golgi ribbon must be "unlinked" into isolated stacks to allow progression into mitosis. Here we show that the Golgi-associated protein GRASP65, which is well known for its role in Golgi stacking and ribbon formation, is also required for the organization of the microtubule cytoskeleton. GRASP65 is not involved in microtubule nucleation or anchoring. Instead, it is required for the stabilization of newly nucleated microtubules, leading to their acetylation and clustering of Golgi stacks. Ribbon formation and microtubule stabilization are both regulated by JNK/ERK-mediated phosphorylation of S274 of GRASP65, suggesting that this protein can coordinate the Golgi structure with microtubule organization. In agreement with an important role, tubulin acetylation is strongly reduced during the G2 phase of the cell cycle, allowing the separation of the Golgi stacks. Thus, our data reveal a fundamental role of GRASP65 in the integration of different stimuli to modulate Golgi structure and microtubule organization during cell division.

Mitotic inheritance of the Golgi complex and its role in cell division.

The Golgi apparatus plays essential roles in the processing and sorting of proteins and lipids, but it can also act as a signalling hub and a microtubule-nucleation centre. The Golgi complex (GC) of mammalian cells is composed of stacks connected by tubular bridges to form a continuous membranous system. In spite of this structural complexity, the GC is highly dynamic, and this feature becomes particularly evident during mitosis, when the GC undergoes a multi-step disassembly process that allows its correct partitioning and inheritance by daughter cells. Strikingly, different steps of Golgi disassembly control mitotic entry and progression, indicating that cells actively monitor Golgi integrity during cell division. Here, we summarise the basic mechanisms and the molecular players that are involved in Golgi disassembly, focussing in particular on recent studies that have revealed the fundamental signalling pathways that connect Golgi inheritance to mitotic entry and progression.

Alterations of Golgi organization in Alzheimer's disease: A cause or a consequence?

The Golgi apparatus is a central organelle of the secretory pathway involved in the post-translational modification and sorting of lipids and proteins. In mammalian cells, the Golgi apparatus is composed of stacks of cisternae organized in polarized manner, which are interconnected by membrane tubules to constitute the Golgi ribbon, located in the proximity of the centrosome. Besides the processing and transport of cargo, the Golgi complex is actively involved in the regulation of mitotic entry, cytoskeleton organization and dynamics, calcium homeostasis, and apoptosis, representing a signalling platform for the control of several cellular functions, including signalling initiated by receptors located at the plasma membrane. Alterations of the conventional Golgi organization are associated to many disorders, such as cancer or different neurodegenerative diseases. In this review, we examine the functional implications of modifications of Golgi structure in neurodegenerative disorders, with a focus on the role of Golgi fragmentation in the development of Alzheimer's disease. The comprehension of the mechanism that induces Golgi fragmentation and of its downstream effects on neuronal function have the potential to contribute to the development of more effective therapies to treat or prevent some of these disorders.

Assays to Study the Fragmentation of the Golgi Complex During the G2-M Transition of the Cell Cycle.

The Golgi complex of mammalian cells is composed of stacks of flattened cisternae that are connected by tubules to form a continuous membrane system, also known as the Golgi ribbon. At the onset of mitosis, the Golgi ribbon is progressively fragmented into small tubular-vesicular clusters and it is reconstituted before completion of cytokinesis. The investigation of the mechanisms behind this reversible cycle of disassembly and reassembly has led to the identification of structural Golgi proteins and regulators. Moreover, these studies allowed to discover that disassembly of the ribbon is necessary for cell entry into mitosis. Here, we describe an in vitro assay that reproduces the mitotic Golgi fragmentation and that has been successfully employed to identify many important mechanisms and proteins involved in the mitotic Golgi reorganization.

Aurora-A recruitment and centrosomal maturation are regulated by a Golgi-activated pool of Src during G2.

The Golgi apparatus is composed of stacks of cisternae laterally connected by tubules to form a ribbon-like structure. At the onset of mitosis, the Golgi ribbon is broken down into discrete stacks, which then undergo further fragmentation. This ribbon cleavage is required for G2/M transition, which thus indicates that a 'Golgi mitotic checkpoint' couples Golgi inheritance with cell cycle transition. We previously showed that the Golgi-checkpoint regulates the centrosomal recruitment of the mitotic kinase Aurora-A; however, how the Golgi unlinking regulates this recruitment was unknown. Here we show that, in G2, Aurora-A recruitment is promoted by activated Src at the Golgi. Our data provide evidence that Src and Aurora-A interact upon Golgi ribbon fragmentation; Src phosphorylates Aurora-A at tyrosine 148 and this specific phosphorylation is required for Aurora-A localization at the centrosomes. This process, pivotal for centrosome maturation, is a fundamental prerequisite for proper spindle formation and chromosome segregation.

JNK2 controls fragmentation of the Golgi complex and the G2/M transition through phosphorylation of GRASP65.

In mammalian cells, the Golgi complex is composed of stacks that are connected by membranous tubules. During G2, the Golgi complex is disassembled into isolated stacks. This process is required for entry into mitosis, indicating that the correct inheritance of the organelle is monitored by a 'Golgi mitotic checkpoint'. However, the regulation and the molecular mechanisms underlying this Golgi disassembly are still poorly understood. Here, we show that JNK2 has a crucial role in the G2-specific separation of the Golgi stacks through phosphorylation of Ser277 of the Golgi-stacking protein GRASP65 (also known as GORASP1). Inhibition of JNK2 by RNA interference or by treatment with three unrelated JNK inhibitors causes a potent and persistent cell cycle block in G2. JNK activity becomes dispensable for mitotic entry if the Golgi complex is disassembled by brefeldin A treatment or by GRASP65 depletion. Finally, measurement of the Golgi fluorescence recovery after photobleaching demonstrates that JNK is required for the cleavage of the tubules connecting Golgi stacks. Our findings reveal that a JNK2-GRASP65 signalling axis has a crucial role in coupling Golgi inheritance and G2/M transition.

The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

Mechanisms and Regulation of the Mitotic Inheritance of the Golgi Complex.

In mammalian cells, the Golgi complex is structured in the form of a continuous membranous system composed of stacks connected by tubular bridges: the "Golgi ribbon." At the onset of mitosis, the Golgi complex undergoes a multi-step fragmentation process that is required for its correct partition into the dividing cells. Importantly, inhibition of Golgi disassembly results in cell-cycle arrest at the G2 stage, which indicates that accurate inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." Moreover, mitotic Golgi disassembly correlates with the release of a set of Golgi-localized proteins that acquire specific functions during mitosis, such as mitotic spindle formation and regulation of the spindle checkpoint. Most of these events are regulated by small GTPases of the Arf and Rab families. Here, we review recent studies that are revealing the fundamental mechanisms, the molecular players, and the biological significance of mitotic inheritance of the Golgi complex in mammalian cells. We also briefly comment on how Golgi partitioning is coordinated with mitotic progression.