New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

The 4th dimension of in vitro systems - Time to level up.

Various in vitro model systems have been established over the last decades to understand physiological processes, the causalities of diseases and the response of humans to environmental and industrial chemicals or therapeutic drugs. Common to all is a limited biological significance due to the impairment of functionality, for instance by the lack of physiological 3D tissue architecture or the loss of fundamental regulatory mechanisms including the circadian rhythm. The circadian rhythm is an adaption of living organisms to rhythmic environmental changes of the day-night cycle and coordinates behavior as well as various crucial physiological processes in a 24-hour pattern. Here, we discuss the impact of integrating circadian regulation in experimental approaches and toxicological assessments to improve the biological relevance of the obtained results. In particular, it is known for some time that an ongoing disruption of the circadian rhythmicity is associated with an increased risk for cardiovascular disease, metabolic dysfunction or cancer. In the context of health recovery, the importance of circadian control mechanism is recognized by chronopharmacological concepts to increase the efficiency of pharmacological treatment strategies. Despite the undeniable circadian dependency and the biological relevance of manifold cellular and molecular processes, the impact of circadian regulation is hardly considered in a wide range of biomedical and toxicological research areas. Reactivating the circadian regulation holds the promise to enhance the biological relevance and reliability of in vitro approaches. In the context of human health protection the implementation of a circadian regulation will subsequently generate advanced physiologically relevant in vitro approaches and allows an improved toxicological assessment of health risks. In addition, the establishment of circadian disruption as a novel toxicological endpoint will provide a better understanding of toxicological mode of actions of environmental and industrial chemicals or drugs and enlarge the knowledge of disease development.

Integration of Epigenetic Mechanisms into Non-Genotoxic Carcinogenicity Hazard Assessment: Focus on DNA Methylation and Histone Modifications.

Epigenetics involves a series of mechanisms that entail histone and DNA covalent modifications and non-coding RNAs, and that collectively contribute to programing cell functions and differentiation. Epigenetic anomalies and DNA mutations are co-drivers of cellular dysfunctions, including carcinogenesis. Alterations of the epigenetic system occur in cancers whether the initial carcinogenic events are from genotoxic (GTxC) or non-genotoxic (NGTxC) carcinogens. NGTxC are not inherently DNA reactive, they do not have a unifying mode of action and as yet there are no regulatory test guidelines addressing mechanisms of NGTxC. To fil this gap, the Test Guideline Programme of the Organisation for Economic Cooperation and Development is developing a framework for an integrated approach for the testing and assessment (IATA) of NGTxC and is considering assays that address key events of cancer hallmarks. Here, with the intent of better understanding the applicability of epigenetic assays in chemical carcinogenicity assessment, we focus on DNA methylation and histone modifications and review: (1) epigenetic mechanisms contributing to carcinogenesis, (2) epigenetic mechanisms altered following exposure to arsenic, nickel, or phenobarbital in order to identify common carcinogen-specific mechanisms, (3) characteristics of a series of epigenetic assay types, and (4) epigenetic assay validation needs in the context of chemical hazard assessment. As a key component of numerous NGTxC mechanisms of action, epigenetic assays included in IATA assay combinations can contribute to improved chemical carcinogen identification for the better protection of public health.

Chemical carcinogen safety testing: OECD expert group international consensus on the development of an integrated approach for the testing and assessment of chemical non-genotoxic carcinogens.

While regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests (which include mutagenicity assays). If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, but under most chemical regulations (except plant protection, biocides, pharmaceuticals), this is rare. The decision to conduct further testing based on genotoxicity test outcomes creates a regulatory gap for the identification of non-genotoxic carcinogens (NGTxC). With the objective of addressing this gap, in 2016, the Organization of Economic Cooperation and Development (OECD) established an expert group to develop an integrated approach to the testing and assessment (IATA) of NGTxC. Through that work, a definition of NGTxC in a regulatory context was agreed. Using the adverse outcome pathway (AOP) concept, various cancer models were developed, and overarching mechanisms and modes of action were identified. After further refining and structuring with respect to the common hallmarks of cancer and knowing that NGTxC act through a large variety of specific mechanisms, with cell proliferation commonly being a unifying element, it became evident that a panel of tests covering multiple biological traits will be needed to populate the IATA. Consequently, in addition to literature and database investigation, the OECD opened a call for relevant assays in 2018 to receive suggestions. Here, we report on the definition of NGTxC, on the development of the overarching NGTxC IATA, and on the development of ranking parameters to evaluate the assays. Ultimately the intent is to select the best scoring assays for integration in an NGTxC IATA to better identify carcinogens and reduce public health hazards.

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.

Restoring circadian synchrony in vitro facilitates physiological responses to environmental chemicals.

BACKGROUND: The growing requirement of hazard and risk assessment of environmental chemicals and the efforts to minimize animal testing, increases the demand for innovative and predictive in vitro test systems in toxicology, reflecting the physiological conditions of human nature. Here, an elemental factor regulating a variety of physiological processes is the day-night rhythm. This circadian rhythm, describing a biological oscillation with a 24-h period is hardly acknowledged in toxicology and test method development. Whilst, in animals or humans the entire organism exhibits a rigorous cellular circadian synchrony, in conventional in vitro systems each cell follows its own rhythm, due to the absence of appropriate synchronizing signals. OBJECTIVE: Here we investigated whether circadian synchronization of human cells in an in vitro system improves the cellular response and, thus, increases the sensitivity of the test system. Since the circadian regulation of metabolism is particularly well understood, and dioxin and dioxin-like compounds are of major concern for environmental health we focused on the ubiquitous drug metabolizing detoxification system mediated by the aryl hydrocarbon receptor (AHR). METHODS: To this end, we applied various prototypical AHR activators onto different human cell lines under non-synchronized or circadian synchronized conditions and determined the dose response on representative endogenous target genes. RESULTS: Remarkably, the cellular response dynamic upon chemical treatment was substantially enhanced in circadian synchronized cells and followed a rhythmic expression pattern. This broader dynamic range was associated with a strikingly higher induction of AHR target genes and the corresponding enzymatic activity, thereby rather mimicking the in vivo situation. CONCLUSION: Our findings indicate that a synchronized circadian rhythm in a cell culture based test system can improve the physiological relevance of an appropriate in vitro method by reflecting the biological in vivo situation more closely. Accordingly, it is a promising tool to facilitate the wide acceptance of in vitro methods in the field of regulatory toxicology and to further optimize the toxicological assessment of environmental chemicals.

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.

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.

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.