Distinct Mitotic Functions of Nucleolar and Spindle-Associated Protein 1 (NuSAP1) Are Controlled by Two Consensus SUMOylation Sites.

Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation. NuSAP1 regulates these processes by interacting with several protein partners. Its abundance, activity and interactions are therefore tightly regulated during mitosis. Protein conjugation with SUMO (Small Ubiquitin-like MOdifier peptide) is a reversible post-translational modification that modulates rapid changes in the structure, interaction(s) and localization of proteins. NuSAP1 was previously found to interact with RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilizing activity, but how this interaction affects NuSAP1 activity has remained elusive. Here, we show that NuSAP1 interacts with RANBP2 and forms proximity ligation products with SUMO2/3 peptides in a RANBP2-dependent manner at key mitotic sites. A bioinformatic search identified two putative SUMO consensus sites in NuSAP1, within the DNA-binding and the microtubule-binding domains, respectively. Site-specific mutagenesis, and mitotic phenotyping in cell lines expressing each NuSAP1 mutant version, revealed selective roles of each individual site in control of NuSAP1 localization and in generation of specific mitotic defects and distinct fates in daughter cells. These results identify therefore two new regulatory sites for NuSAP1 functions and implicate RANBP2 in control of NuSAP1 activity.

Aurora B SUMOylation Is Restricted to Centromeres in Early Mitosis and Requires RANBP2.

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.

RS6077 induces mitotic arrest and selectively activates cell death in human cancer cell lines and in a lymphoma tumor in vivo.

We synthesized a new inhibitor of tubulin polymerization, the pyrrole (1-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrrol-3-yl)(3,4,5-trimethoxy-phenyl)methanone 6 (RS6077). Compound 6 inhibited the growth of multiple cancer cell lines, with IC50 values in the nM range, without affecting the growth of non-transformed cells. The novel agent arrested cells in the G2/M phase of the cell cycle in both transformed and non-transformed cell lines, but single cell analysis by time-lapse video recording revealed a remarkable selectivity in cell death induction by compound 6: in RPE-1 non-transformed cells mitotic arrest induced was not necessarily followed by cell death; in contrast, in HeLa transformed and in lymphoid-derived transformed AHH1 cell lines, cell death was effectively induced during mitotic arrest in cells that fail to complete mitosis. Importantly, the agent also inhibited the growth of the lymphoma TMD8 xenograft model. Together these findings suggest that derivative 6 has a selective efficacy in transformed vs non-transformed cells and indicate that the same compound has potential as novel therapeutic agent to treat lymphomas. Compound 6 showed good metabolic stability upon incubation with human liver microsomes.

Pharmacological targeting of CBP/p300 drives a redox/autophagy axis leading to senescence-induced growth arrest in non-small cell lung cancer cells.

p300/CBP histone acetyltransferases (HAT) are critical transcription coactivators involved in multiple cellular activities. They act at multiple levels in non-small cell lung carcinoma (NSCLC) and appear, therefore, as promising druggable targets. Herein, we investigated the biological effects of A-485, the first selective (potent) drug-like HAT catalytic inhibitor of p300/CBP, in human NSCLC cell lines. A-485 treatment specifically reduced p300/CBP-mediated histone acetylation marks and caused growth arrest of lung cancer cells via activation of the autophagic pathway. Indeed, A-485 growth-arrested cells displayed phenotypic markers of cell senescence and failed to form colonies. Notably, disruption of autophagy by genetic and pharmacological approaches triggered apoptotic cell death. Mechanistically, A-485-induced senescence occurred through the accumulation of reactive oxygen species (ROS), which in turn resulted in DNA damage and activation of the autophagic pathway. Interestingly, ROS scavengers were able to revert senescence phenotype and restore cell viability, suggesting that ROS production had a key role in upstream events leading to growth arrest commitment. Altogether, our data provide new insights into the biological effects of the A-485 and uncover the importance of the autophagic/apoptotic response to design a new combinatorial anticancer strategy.

An Epigenetic LINE-1-Based Mechanism in Cancer.

In the last fifty years, large efforts have been deployed in basic research, clinical oncology, and clinical trials, yielding an enormous amount of information regarding the molecular mechanisms of cancer and the design of effective therapies. The knowledge that has accumulated underpins the complexity, multifactoriality, and heterogeneity of cancer, disclosing novel landscapes in cancer biology with a key role of genome plasticity. Here, we propose that cancer onset and progression are determined by a stress-responsive epigenetic mechanism, resulting from the convergence of upregulation of LINE-1 (long interspersed nuclear element 1), the largest family of human retrotransposons, genome damage, nuclear lamina fragmentation, chromatin remodeling, genome reprogramming, and autophagy activation. The upregulated expression of LINE-1 retrotransposons and their protein products plays a key role in these processes, yielding an increased plasticity of the nuclear architecture with the ensuing reprogramming of global gene expression, including the reactivation of embryonic transcription profiles. Cancer phenotypes would thus emerge as a consequence of the unscheduled reactivation of embryonic gene expression patterns in an inappropriate context, triggering de-differentiation and aberrant proliferation in differentiated cells. Depending on the intensity of the stressing stimuli and the level of LINE-1 response, diverse degrees of malignity would be generated.

Non-transport roles of nuclear import receptors: In need of the right balance.

Nuclear import receptors ensure the recognition and transport of proteins across the nuclear envelope into the nucleus. In addition, as diverse processes as mitosis, post-translational modifications at mitotic exit, ciliogenesis, and phase separation, all share a common need for regulation by nuclear import receptors - particularly importin beta-1 and importin beta-2/transportin - independent on nuclear import. In particular, 1) nuclear import receptors regulate the mitotic spindle after nuclear envelope breakdown, 2) they shield cargoes from unscheduled ubiquitination, regulating their timely proteolysis; 3) they regulate ciliary factors, crucial to cell communications and tissue architecture during development; and 4) they prevent phase separation of toxic proteins aggregates in neurons. The balance of nuclear import receptors to cargoes is critical in all these processes, albeit in opposite directions: overexpression of import receptors, as often found in cancer, inhibits cargoes and impairs downstream processes, motivating the therapeutic design of specific inhibitors. On the contrary, elevated expression is beneficial in neuronal contexts, where nuclear import receptors are regarded as potential therapeutic tools in counteracting the formation of aggregates that may cause neurodegeneration. This paradox demonstrates the amplitude of nuclear import receptors-dependent functions in different contexts and adds complexity in considering their therapeutic implications.

Circular RNA ZNF609/CKAP5 mRNA interaction regulates microtubule dynamics and tumorigenicity.

Circular RNAs (circRNAs) are widely expressed in eukaryotes and are regulated in many biological processes. Although several studies indicate their activity as microRNA (miRNA) and protein sponges, little is known about their ability to directly control mRNA homeostasis. We show that the widely expressed circZNF609 directly interacts with several mRNAs and increases their stability and/or translation by favoring the recruitment of the RNA-binding protein ELAVL1. Particularly, the interaction with CKAP5 mRNA, which interestingly overlaps the back-splicing junction, enhances CKAP5 translation, regulating microtubule function in cancer cells and sustaining cell-cycle progression. Finally, we show that circZNF609 downregulation increases the sensitivity of several cancer cell lines to different microtubule-targeting chemotherapeutic drugs and that locked nucleic acid (LNA) protectors against the pairing region on circZNF609 phenocopy such effects. These data set an example of how the small effects tuned by circZNF609/CKAP5 mRNA interaction might have a potent output in tumor growth and drug response.

Reverse transcriptase inhibitors promote the remodelling of nuclear architecture and induce autophagy in prostate cancer cells.

Emerging data indicate that the reverse transcriptase (RT) protein encoded by LINE-1 transposable elements is a promising cancer target. Nonnucleoside RT inhibitors, e.g. efavirenz (EFV) and SPV122.2, reduce proliferation and promote differentiation of cancer cells, concomitant with a global reprogramming of the transcription profile. Both inhibitors have therapeutic anticancer efficacy in animal models. Here we have sought to clarify the mechanisms of RT inhibitors in cancer cells. We report that exposure of PC3 metastatic prostate carcinoma cells to both RT inhibitors results in decreased proliferation, and concomitantly induces genome damage. This is associated with rearrangements of the nuclear architecture, particularly at peripheral chromatin, disruption of the nuclear lamina, and budding of micronuclei. These changes are reversible upon discontinuation of the RT-inhibitory treatment, with reconsititution of the lamina and resumption of the cancer cell original features. The use of pharmacological autophagy inhibitors proves that autophagy is largely responsible for the antiproliferative effect of RT inhibitors. These alterations are not induced in non-cancer cell lines exposed to RT inhibitors. These data provide novel insight in the molecular pathways targeted by RT inhibitors in cancer cells.

Importin-ß/karyopherin-ß1 modulates mitotic microtubule function and taxane sensitivity in cancer cells via its nucleoporin-binding region.

The nuclear transport receptor importin-ß/karyopherin-ß1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-ß regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-ß's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-ß form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-ß affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-ß interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-ß, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-ß's nucleoporin-binding residues. Concomitantly, importin-ß overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-ß function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.

The Mitotic Apparatus and Kinetochores in Microcephaly and Neurodevelopmental Diseases.

Regulators of mitotic division, when dysfunctional or expressed in a deregulated manner (over- or underexpressed) in somatic cells, cause chromosome instability, which is a predisposing condition to cancer that is associated with unrestricted proliferation. Genes encoding mitotic regulators are growingly implicated in neurodevelopmental diseases. Here, we briefly summarize existing knowledge on how microcephaly-related mitotic genes operate in the control of chromosome segregation during mitosis in somatic cells, with a special focus on the role of kinetochore factors. Then, we review evidence implicating mitotic apparatus- and kinetochore-resident factors in the origin of congenital microcephaly. We discuss data emerging from these works, which suggest a critical role of correct mitotic division in controlling neuronal cell proliferation and shaping the architecture of the central nervous system.

New 6- and 7-heterocyclyl-1H-indole derivatives as potent tubulin assembly and cancer cell growth inhibitors.

We designed new 3-arylthio- and 3-aroyl-1H-indole derivatives 3-22 bearing a heterocyclic ring at position 5, 6 or 7 of the indole nucleus. The 6- and 7-heterocyclyl-1H-indoles showed potent inhibition of tubulin polymerization, binding of colchicine to tubulin and growth of MCF-7 cancer cells. Compounds 13 and 19 inhibited a panel of cancer cells and the NCI/ADR-RES multidrug resistant cell line at low nanomolar concentrations. Compound 13 at 50 nM induced 77% G2/M in HeLa cells, and at 20 nM caused 50% stable arrest of mitosis. As an inhibitor of HepG2 cells (IC50 = 20 nM), 13 was 4-fold superior to 19. Compound 13 was a potent inhibitor of the human U87MG glioblastoma cells at nanomolar concentrations, being nearly one order of magnitude superior to previously reported arylthioindoles. The present results highlight 13 as a robust scaffold for the design of new anticancer agents.

Visualization of human karyopherin beta-1/importin beta-1 interactions with protein partners in mitotic cells by co-immunoprecipitation and proximity ligation assays.

Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells. By combining co-immunoprecipitation and proteome-wide mass spectrometry analysis of synchronized cell extracts, we identified expected (e.g., RAN and SUMO pathway factors) and novel mitotic interactors of importin beta-1, many with RNA-binding ability, that had not been previously associated with importin beta-1. These data complement interactomic studies of interphase transport pathways. We further developed automated proximity ligation assay (PLA) protocols to validate selected interactors. We succeeded in obtaining spatial and temporal resolution of genuine importin beta-1 interactions, which were visualized and localized in situ in intact mitotic cells. Further developments of PLA protocols will be helpful to dissect importin beta-1-orchestrated pathways during mitosis.

Subcellular localization of the five members of the human steroid 5α-reductase family.

In humans the steroid 5alpha-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

Importin-ß and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function.

Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2-SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-ß and CRM1 play essential roles in localising the RANBP2-SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO-conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2.

Identification of small molecule inhibitors of the Aurora-A/TPX2 complex.

Aurora kinases are a family of cell division regulators that govern the correct assembly of a bipolar mitotic spindle and the fidelity of chromosome segregation. Their overexpression is associated with genomic instability and aneuploidy, and is frequently observed in cancer. Accordingly, competitive inhibitors targeting Aurora kinase activity at the ATP-binding site are being investigated for therapeutic purposes. Despite promising pre-clinical data, these molecules display moderate effects in clinical trials and incomplete selectivity, either against distinct family members, or other kinases. As an alternative approach, protein-protein interaction inhibitors targeting mitotic kinases and their activators can be exploited to achieve increased specificity of action. In this study, a virtual screening of small molecules led to the identification of 25 potential inhibitors of the interaction between Aurora-A and its activator TPX2. In vitro experiments confirmed that 4 hits bind Aurora-A in the low micromolar range and compete for TPX2 binding. Immunofluorescence assays showed that 2 compounds also yield lowered Aurora-A activity and spindle pole defects in cultured osteosarcoma cells. The identified protein-protein interaction inhibitors of the Aurora-A/TPX2 complex might represent lead compounds for further development towards pioneering anti-cancer drugs and provide the proof-of-concept for a new exploitable strategy to target mitotic kinases.

Mitotic cell death induction by targeting the mitotic spindle with tubulin-inhibitory indole derivative molecules.

Tubulin-targeting molecules are widely used cancer therapeutic agents. They inhibit microtubule-based structures, including the mitotic spindle, ultimately preventing cell division. The final fates of microtubule-inhibited cells are however often heterogeneous and difficult to predict. While recent work has provided insight into the cell response to inhibitors of microtubule dynamics (taxanes), the cell response to tubulin polymerization inhibitors remains less well characterized. Arylthioindoles (ATIs) are recently developed tubulin inhibitors. We previously identified ATI members that effectively inhibit tubulin polymerization in vitro and cancer cell growth in bulk cell viability assays. Here we characterise in depth the response of cancer cell lines to five selected ATIs. We find that all ATIs arrest mitotic progression, yet subsequently yield distinct cell fate profiles in time-lapse recording assays, indicating that molecules endowed with similar tubulin polymerization inhibitory activity in vitro can in fact display differential efficacy in living cells. Individual ATIs induce cytological phenotypes of increasing severity in terms of damage to the mitotic apparatus. That differentially triggers MCL-1 down-regulation and caspase-3 activation, and underlies the terminal fate of treated cells. Collectively, these results contribute to define the cell response to tubulin inhibitors and pinpoint potentially valuable molecules that can increase the molecular diversity of tubulin-targeting agents.

p38 MAPK differentially controls NK activating ligands at transcriptional and post-transcriptional level on multiple myeloma cells.

The mechanisms that regulate the expression of the NKG2D and DNAM-1 activating ligands are only partially known, but it is now widely established that their expression is finely regulated at transcriptional, post-transcriptional and post-translational level, and involve numerous stress pathways depending on the type of ligand, stressor, and cell context. We show that treatment of Multiple Myeloma (MM) cells with sub-lethal doses of Vincristine (VCR), an anticancer drug that inhibits the assembly of microtubules, stimulates the expression of NKG2D and DNAM-1 activating ligands, rendering these cells more susceptible to NK cell-mediated killing. Herein, we focused our attention on the identification of the signaling pathways leading to de novo surface expression of ULBP-1, and to MICA and PVR upregulation on VCR-treated MM cells, both at protein and mRNA levels. We found that p38MAPK differentially regulates drug-dependent ligand upregulation at transcriptional and post-transcriptional level. More specifically, we observed that ULBP-1 expression is attributable to both increased transcriptional activity mediated by ATM-dependent p53 activation, and enhanced mRNA stability; while the p38-activated E2F1 transcription factor regulates MICA and PVR mRNA expression. All together, our findings reveal a previously unrecognized activity of VCR as anticancer agent, and indicate that in addition to its established ability to arrest cell growth, VCR can also modulate the expression of NKG2D and DNAM-1 activating ligand on tumor cells and thus promoting NK cell-mediated immunosurveillance.

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

Targeting nuclear transporters in cancer: Diagnostic, prognostic and therapeutic potential.

The Karyopherin superfamily is a major class of soluble transport receptors consisting of both import and export proteins. The trafficking of proteins involved in transcription, cell signalling and cell cycle regulation among other functions across the nuclear membrane is essential for normal cellular functioning. However, in cancer cells, the altered expression or localization of nuclear transporters as well as the disruption of endogenous nuclear transport inhibitors are some ways in which the Karyopherin proteins are dysregulated. The value of nuclear transporters in the diagnosis, prognosis and treatment of cancer is currently being elucidated with recent studies highlighting their potential as biomarkers and therapeutic targets.

The GTPase RAN regulates multiple steps of the centrosome life cycle.

Growing lines of evidence implicate the small GTPase RAN, its regulators and effectors--predominantly, nuclear transport receptors--in practically all aspects of centrosome biology in mammalian cells. These include duplication licensing, cohesion, positioning, and microtubule-nucleation capacity. RAN cooperates with the protein nuclear export vector exportin 1/CRM1 to recruit scaffolding proteins containing nuclear export sequences that play roles in the structural organization of centrosomes. Together, they also limit centrosome reduplication by regulating the localization of key "licensing" proteins during the centrosome duplication cycle. In parallel, RAN also regulates the capacity of centrosomes to nucleate and organize functional microtubules, and this predominanlty involves importin vectors: many factors regulating microtubule nucleation or function harbor nuclear localization sequences that interact with importin molecules and such interaction inhibits their activity. Active RANGTP binding to importin molecules removes the inhibition and releases microtubule regulatory factors in the free productive form. A dynamic scenario emerges, in which RAN is pivotal in linking spatiotemporal control of centrosome regulators to the cell cycle machinery.