Automatic classification of experimental models in biomedical literature to support searching for alternative methods to animal experiments.

Current animal protection laws require replacement of animal experiments with alternative methods, whenever such methods are suitable to reach the intended scientific objective. However, searching for alternative methods in the scientific literature is a time-consuming task that requires careful screening of an enormously large number of experimental biomedical publications. The identification of potentially relevant methods, e.g. organ or cell culture models, or computer simulations, can be supported with text mining tools specifically built for this purpose. Such tools are trained (or fine tuned) on relevant data sets labeled by human experts. We developed the GoldHamster corpus, composed of 1,600 PubMed (Medline) articles (titles and abstracts), in which we manually identified the used experimental model according to a set of eight labels, namely: "in vivo", "organs", "primary cells", "immortal cell lines", "invertebrates", "humans", "in silico" and "other" (models). We recruited 13 annotators with expertise in the biomedical domain and assigned each article to two individuals. Four additional rounds of annotation aimed at improving the quality of the annotations with disagreements in the first round. Furthermore, we conducted various machine learning experiments based on supervised learning to evaluate the corpus for our classification task. We obtained more than 7,000 document-level annotations for the above labels. After the first round of annotation, the inter-annotator agreement (kappa coefficient) varied among labels, and ranged from 0.42 (for "others") to 0.82 (for "invertebrates"), with an overall score of 0.62. All disagreements were resolved in the subsequent rounds of annotation. The best-performing machine learning experiment used the PubMedBERT pre-trained model with fine-tuning to our corpus, which gained an overall f-score of 0.83. We obtained a corpus with high agreement for all labels, and our evaluation demonstrated that our corpus is suitable for training reliable predictive models for automatic classification of biomedical literature according to the used experimental models. Our SMAFIRA - "Smart feature-based interactive" - search tool ( https://smafira.bf3r.de ) will employ this classifier for supporting the retrieval of alternative methods to animal experiments. The corpus is available for download ( https://doi.org/10.5281/zenodo.7152295 ), as well as the source code ( https://github.com/mariananeves/goldhamster ) and the model ( https://huggingface.co/SMAFIRA/goldhamster ).

GPER1 links estrogens to centrosome amplification and chromosomal instability in human colon cells.

The role of the alternate G protein-coupled estrogen receptor 1 (GPER1) in colorectal cancer (CRC) development and progression is unclear, not least because of conflicting clinical and experimental evidence for pro- and anti-tumorigenic activities. Here, we show that low concentrations of the estrogenic GPER1 ligands, 17ß-estradiol, bisphenol A, and diethylstilbestrol cause the generation of lagging chromosomes in normal colon and CRC cell lines, which manifest in whole chromosomal instability and aneuploidy. Mechanistically, (xeno)estrogens triggered centrosome amplification by inducing centriole overduplication that leads to transient multipolar mitotic spindles, chromosome alignment defects, and mitotic laggards. Remarkably, we could demonstrate a significant role of estrogen-activated GPER1 in centrosome amplification and increased karyotype variability. Indeed, both gene-specific knockdown and inhibition of GPER1 effectively restored normal centrosome numbers and karyotype stability in cells exposed to 17ß-estradiol, bisphenol A, or diethylstilbestrol. Thus, our results reveal a novel link between estrogen-activated GPER1 and the induction of key CRC-prone lesions, supporting a pivotal role of the alternate estrogen receptor in colon neoplastic transformation and tumor progression.

Estrogens-Origin of Centrosome Defects in Human Cancer?

Estrogens are associated with a variety of diseases and play important roles in tumor development and progression. Centrosome defects are hallmarks of human cancers and contribute to ongoing chromosome missegragation and aneuploidy that manifest in genomic instability and tumor progression. Although several mechanisms underlie the etiology of centrosome aberrations in human cancer, upstream regulators are hardly known. Accumulating experimental and clinical evidence points to an important role of estrogens in deregulating centrosome homeostasis and promoting karyotype instability. Here, we will summarize existing literature of how natural and synthetic estrogens might contribute to structural and numerical centrosome defects, genomic instability and human carcinogenesis.

Biomedical Research Meets Toxicology: How In Vitro Chromosome Instability Methods Can Contribute to Carcinogenicity Prediction.

Cancer is a major health concern and a leading cause of mortality. The reliable identification of carcinogens and understanding of carcinogenicity has become a main focus of biomedical research and regulatory toxicology. While biomedical research applies cellular in vitro methods to uncover the underlying mechanisms causing cancer, regulatory toxicology relies on animal testing to predict carcinogenicity of chemicals, often with limited human relevance. Exemplified by chromosome instability-mediated carcinogenicity, we discuss the need to combine the strengths of both fields to develop highly predictive and mechanism-derived in vitro methods that facilitate risk assessment in respect to relevant human diseases.

Endocrine Disruptors: Adverse Health Effects Mediated by EGFR?

Although endocrine disruptors represent a serious concern to human health, the underlying molecular mechanisms leading to diseases such as cancer remain poorly understood. Recent work has uncovered the epidermal growth factor receptor (EGFR) as a possible mediator of these adverse health effects, with important implications for the role of endocrine disruptors in human diseases.

Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data.

To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery.

CHK2-BRCA1 tumor-suppressor axis restrains oncogenic Aurora-A kinase to ensure proper mitotic microtubule assembly.

BRCA1 (breast cancer type 1 susceptibility protein) is a multifunctional tumor suppressor involved in DNA damage response, DNA repair, chromatin regulation, and mitotic chromosome segregation. Although the nuclear functions of BRCA1 have been investigated in detail, its role during mitosis is little understood. It is clear, however, that loss of BRCA1 in human cancer cells leads to chromosomal instability (CIN), which is defined as a perpetual gain or loss of whole chromosomes during mitosis. Moreover, our recent work has revealed that the mitotic function of BRCA1 depends on its phosphorylation by the tumor-suppressor kinase Chk2 (checkpoint kinase 2) and that this regulation is required to ensure normal microtubule plus end assembly rates within mitotic spindles. Intriguingly, loss of the positive regulation of BRCA1 leads to increased oncogenic Aurora-A activity, which acts as a mediator for abnormal mitotic microtubule assembly resulting in chromosome missegregation and CIN. However, how the CHK2-BRCA1 tumor suppressor axis restrains oncogenic Aurora-A during mitosis to ensure karyotype stability remained an open question. Here we uncover a dual molecular mechanism by which the CHK2-BRCA1 axis restrains oncogenic Aurora-A activity during mitosis and identify BRCA1 itself as a target for Aurora-A relevant for CIN. In fact, Chk2-mediated phosphorylation of BRCA1 is required to recruit the PP6C-SAPS3 phosphatase, which acts as a T-loop phosphatase inhibiting Aurora-A bound to BRCA1. Consequently, loss of CHK2 or PP6C-SAPS3 promotes Aurora-A activity associated with BRCA1 in mitosis. Aurora-A, in turn, then phosphorylates BRCA1 itself, thereby inhibiting the mitotic function of BRCA1 and promoting mitotic microtubule assembly, chromosome missegregation, and CIN.

Fresh WNT into the regulation of mitosis.

Canonical Wnt signaling triggering ß-catenin-dependent gene expression contributes to cell cycle progression, in particular at the G1/S transition. Recently, however, it became clear that the cell cycle can also feed back on Wnt signaling at the G2/M transition. This is illustrated by the fact that mitosis-specific cyclin-dependent kinases can phosphorylate the Wnt co-receptor LRP6 to prime the pathway for incoming Wnt signals when cells enter mitosis. In addition, there is accumulating evidence that various Wnt pathway components might exert additional, Wnt-independent functions that are important for proper regulation of mitosis. The importance of Wnt pathways during mitosis was most recently enforced by the discovery of Wnt signaling contributing to the stabilization of proteins other than ß-catenin, specifically at G2/M and during mitosis. This Wnt-mediated stabilization of proteins, now referred to as Wnt/STOP, might on one hand contribute to maintaining a critical cell size required for cell division and, on the other hand, for the faithful execution of mitosis itself. In fact, most recently we have shown that Wnt/STOP is required for ensuring proper microtubule dynamics within mitotic spindles, which is pivotal for accurate chromosome segregation and for the maintenance of euploidy.

Wnt-mediated protein stabilization ensures proper mitotic microtubule assembly and chromosome segregation.

Wnt signaling stimulates cell proliferation by promoting the G1/S transition of the cell cycle through ß-catenin/TCF4-mediated gene transcription. However, Wnt signaling peaks in mitosis and contributes to the stabilization of proteins other than ß-catenin, a pathway recently introduced as Wnt-dependent stabilization of proteins (Wnt/STOP). Here, we show that Wnt/STOP regulated by basal Wnt signaling during a normal cell cycle is required for proper spindle microtubule assembly and for faithful chromosome segregation during mitosis. Consequently, inhibition of basal Wnt signaling results in increased microtubule assembly rates, abnormal mitotic spindle formation and the induction of aneuploidy in human somatic cells.

A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle orientation.

Proper regulation of microtubule dynamics during mitosis is essential for faithful chromosome segregation. In fact, recently we discovered increased microtubule plus end assembly rates that are frequently observed in human cancer cells as an important mechanism leading to whole chromosome missegregation and chromosomal instability (CIN). However, the genetic alterations responsible for increased microtubule polymerization rates in cancer cells remain largely unknown. The identification of such lesions is hampered by the fact that determining dynamic parameters of microtubules usually involves analyses of living cells, which is technically difficult to perform in large-scale screening settings. Therefore, we sought to identify alternative options to systematically identify regulators of microtubule plus end polymerization. Here, we introduce a simple and robust phenotypic screening assay that is based on the analyses of monopolar mitotic spindle structures that are induced upon inhibition of the mitotic kinesin Eg5/KIF11. We show that increased microtubule polymerization causes highly asymmetric monoasters in the presence of Eg5/KIF11 inhibition and this phenotype can be reliably assessed in living as well as in fixed cells. Using this assay we performed a siRNA screen, in which we identify several microtubule plus end binding proteins as well as centrosomal and cortex associated proteins as important regulators of microtubule plus end assembly. Interestingly, we demonstrate that a subgroup of these regulators function in the regulation of spindle orientation through their role in dampening microtubule plus end polymerization.

Microtubule plus tips: A dynamic route to chromosomal instability.

Although chromosomal instability (CIN) is a recognized hallmark of cancer the underlying mechanisms and consequences are largely unknown. However, it is accepted that lagging chromosomes represent a major prerequisite for chromosome missegregation in cancer cells. Here, we discuss how lagging chromosomes are generated and our recent findings establishing increased microtubule assembly rates as a source of CIN.

Increased microtubule assembly rates influence chromosomal instability in colorectal cancer cells.

Chromosomal instability (CIN) is defined as the perpetual missegregation of whole chromosomes during mitosis and represents a hallmark of human cancer. However, the mechanisms influencing CIN and its consequences on tumour growth are largely unknown. We identified an increase in microtubule plus-end assembly rates as a mechanism influencing CIN in colorectal cancer cells. This phenotype is induced by overexpression of the oncogene AURKA or by loss of the tumour suppressor gene CHK2, a genetic constitution found in 73% of human colorectal cancers. Increased microtubule assembly rates are associated with transient abnormalities in mitotic spindle geometry promoting the generation of lagging chromosomes and influencing CIN. Reconstitution of proper microtubule assembly rates by chemical or genetic means suppresses CIN and thereby, unexpectedly, accelerates tumour growth in vitro and in vivo. Thus, we identify a fundamental mechanism influencing CIN in cancer cells and reveal its adverse consequence on tumour growth.

Tumor suppressor CHK2: regulator of DNA damage response and mediator of chromosomal stability.

CHK2 is a multiorgan tumor susceptibility gene that encodes for a serine/threonine protein kinase involved in the response to cellular DNA damage. After ATM-mediated phosphorylation, the activated Chk2 kinase can act as a signal transducer and phosphorylate a variety of substrates, including the Cdc25 phosphatases, p53, PML, E2F-1, and Brca1, which has been associated with halting the cell cycle, the initiation of DNA repair, and the induction of apoptosis after DNA damage. In addition, recent work has revealed another, DNA-damage-independent function of Chk2 during mitosis that is required for proper mitotic spindle assembly and maintenance of chromosomal stability. This novel role involves a mitotic phosphorylation of the tumor suppressor Brca1 by the Chk2 kinase. On the basis of its role during DNA damage response, Chk2 has been suggested as an anticancer therapy target, but given its recently discovered new function and its role as a tumor suppressor, it is questionable whether inhibition of Chk2 is indeed beneficial for anticancer treatment. However, investigators may be able to exploit the loss of CHK2 in human tumors to develop novel therapies based on synthetic lethal interactions.

Loss of the tumour-suppressor genes CHK2 and BRCA1 results in chromosomal instability.

CHK2 (checkpoint kinase 2) and BRCA1 (breast cancer early-onset 1) are tumour-suppressor genes that have been implicated previously in the DNA damage response. Recently, we have identified CHK2 and BRCA1 as genes required for the maintenance of chromosomal stability and have shown that a Chk2-mediated phosphorylation of Brca1 is required for the proper and timely assembly of mitotic spindles. Loss of CHK2, BRCA1 or inhibition of its Chk2-mediated phosphorylation inevitably results in the transient formation of abnormal spindles that facilitate the establishment of faulty microtubule-kinetochore attachments associated with the generation of lagging chromosomes. Importantly, both CHK2 and BRCA1 are lost at very high frequency in aneuploid lung adenocarcinomas that are typically induced in knockout mice exhibiting chromosomal instability. Thus these results suggest novel roles for Chk2 and Brca1 in mitosis that might contribute to their tumour-suppressor functions.

The CHK2-BRCA1 tumour suppressor pathway ensures chromosomal stability in human somatic cells.

Chromosomal instability (CIN) is a major hallmark of human cancer and might contribute to tumorigenesis. Genes required for the normal progression of mitosis represent potential CIN genes and, as such, are important tumour suppressors. The Chk2 kinase and its downstream targets p53 and Brca1 are tumour suppressors that have been functionally linked to the DNA damage response pathway. Here, we report a function of Chk2, independent of p53 and DNA damage, that is required for proper progression of mitosis, and for the maintenance of chromosomal stability in human somatic cells. Depletion of Chk2 or abrogation of its kinase activity causes abnormal mitotic spindle assembly associated with a delay in mitosis, which promotes the generation of lagging chromosomes, chromosome missegregation and CIN, while still allowing survival and growth. Furthermore, we have identified Brca1 as a mitotic target of the Chk2 kinase in the absence of DNA damage. Accordingly, loss of BRCA1 or its Chk2-mediated phosphorylation leads to spindle formation defects and CIN. Thus, the CHK2-BRCA1 tumour suppressor pathway is required for chromosomal stability, which might contribute to their tumour suppressor function.

Centromere localization of INCENP-Aurora B is sufficient to support spindle checkpoint function.

During mitosis, the chromosomal passenger complex (CPC) comprising the Aurora B kinase, INCENP, survivin and borealin is essential for correcting non-bipolar chromosome attachments and for cytokinesis. In addition, the CPC might fullfil a role in the mitotic spindle assembly checkpoint (SAC), but this activity might be related to its role in correcting non-bipolar chromosome attachments. Here, we demonstrate that treatment of mitotic cells with the antibiotic actinomycin D causes a displacement of an intact and active CPC from centromeres onto chromosome arms, which results in chromosome misalignment, cytokinesis failure and SAC override, but still preserves histone H3 phosphorylation on chromosome arms. This surprising and unique scenario allows the reconstitution of endogenous Aurora B at centromeres/inner kinetochores by expressing a Cenp-B-INCENP fusion protein. We find that although the selective recruitment of endogenous Aurora B to centromeres/inner kinetochores is not sufficient to restore chromosome alignment and cytokinesis, it can restore Cenp-A phosphorylation at kinetochores, BubR1 recruitment to kinetochores and SAC activity after spindle disruption. These results indicate that INCENP-Aurora B localized at centromeres/inner kinetochores is sufficient to mediate SAC activity upon spindle disruption.

Determinants for the efficiency of anticancer drugs targeting either Aurora-A or Aurora-B kinases in human colon carcinoma cells.

The mitotic Aurora kinases, including Aurora-A and Aurora- B, are attractive novel targets for anticancer therapy, and inhibitory drugs have been developed that are currently undergoing clinical trials. However, the molecular mechanisms how these drugs induce tumor cell death are poorly understood. We have addressed this question by comparing the requirements for an efficient induction of apoptosis in response to MLN8054, a selective inhibitor of Aurora-A, and the selective Aurora-B inhibitor ZM447439 in human colon carcinoma cells. By using various isogenic knockout as well as inducible colon carcinoma cell lines, we found that treatment with MLN8054 induces defects in mitotic spindle assembly, which causes a transient spindle checkpoint-dependent mitotic arrest. This cell cycle arrest is not maintained due to the activity of MLN8054 to override the spindle checkpoint. Subsequently, MLN8054-treated cells exit from mitosis and activate a p53-dependent postmitotic G(1) checkpoint, which subsequently induces p21 and Bax, leading to G(1) arrest followed by the induction of apoptosis. In contrast, inhibition of Aurora-B by ZM447439 also interferes with normal chromosome alignment during mitosis and overrides the mitotic spindle checkpoint but allows a subsequent endoreduplication, although ZM447439 potently activates the p53-dependent postmitotic G(1) checkpoint. Moreover, the ZM447439-induced endoreduplication is a prerequisite for the efficiency of the drug. Thus, our results obtained in human colon carcinoma cells indicate that although both Aurora kinase inhibitors are potent inducers of tumor cell death, the pathways leading to the induction of apoptosis in response to these drugs are distinct.

Pharmacologic abrogation of the mitotic spindle checkpoint by an indolocarbazole discovered by cellular screening efficiently kills cancer cells.

The mitotic spindle checkpoint represents a signal transduction pathway that prevents the onset of anaphase until all chromosomes are properly aligned on a metaphase plate. Partial inactivation of this checkpoint allows premature separation of sister chromatids and results in aneuploidy, which might contribute to tumorigenesis. Unlike other cell cycle checkpoints, the spindle checkpoint is essential for cell viability, giving rise to the idea that the spindle checkpoint itself might represent a valuable target for anticancer therapy. We used a cell-based screen and identified the indolocarbazole compound Gö6976 as a pharmacologic inhibitor of the spindle checkpoint. Gö6976 potently overrides a spindle checkpoint-mediated mitotic arrest by abrogating the phosphorylation and kinetochore localization of several spindle checkpoint proteins. We identified the Aurora-A and Aurora-B kinases, which have been previously implicated in proper mitotic progression and spindle checkpoint function, as targets for Gö6976. Accordingly, Gö6976 treatment causes severe mitotic abnormalities and chromosome alignment defects, which are not properly detected by the drug-inactivated spindle checkpoint. This results in an aberrant progression of mitosis, leading to apoptosis in various human cancer cell lines, including spindle checkpoint-compromised cancer cells. Thus, our work describes a novel and promising strategy for anticancer treatment that targets the mitotic spindle checkpoint.