Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - PLOIDYNET (The impact of chromosomal instability on health: Molecular causes and consequences of aneuploidy)

Chromosomal instability (CIN) has been an appreciated hallmark of cancer cells for over a century. Despite this long-standing knowledge, we still do not fully understand how CIN makes cells adopt a malignant fate, or what effect (beneficial/adverse) CIN has on non-transformed cells. Understanding this is important, as it will take us closer to therapies that exploit CIN as a feature that discriminates cancer cells from non-cancer cells, thus yielding better-targeted cancer therapies. To address these questions, a multidisciplinary approach is needed, which was the reason to start PloidyNet, a consortium of academic and industrial investigators, all with an interest in chromosomal instability, and all with a unique research angle.

PloidyNet’s main goal is to train 12 young scientists, 9 ESRs and 3 ERs, in the field of chromosomal instability. To achieve this, we brought together some of Europe’s leading scientists to train our fellows. Within PloidyNet, fellows are thus exposed to a wide variety of state of the art technologies and use several model systems to address how CIN emerges in cancer, to explore what the consequences of CIN are and to find ways of exploiting CIN to improve cancer therapy. PloidyNet consists of 11 beneficiaries and 1 associated partner representing 4 member states and Switzerland including 3 commercial enterprises and training 12 students of 10 nationalities. The majority of our students are female (9 out 11).
To achieve the best possible training, we designed a high quality and highly complementary training program consisting of [1] training by doing research in at least two different labs, [2] industrial rotations and [3] a number of focused workshops, organized by the academic and industrial participants of the network. Our training program captures the multidisciplinary nature of PloidyNet’s participants while providing highly specialized training, as students all have an individual research project that exploits expertise of at least 2-3 participating labs. All students will visit at least one other lab and one of our industrial partners as part of their project. Furthermore, trough Annual Network Meetings, focused courses organized at our hosting institutes, through an interactive WebPortal and a strong social network, students have access to a wide variety of model systems, technologies and expertise throughout their training, which will yield a next generation of scientists that are well prepared for a career in life sciences, either in industry or academia.

In the first two years of PloidyNet, we invested ample time to recruit the best possible students (Y1). In parallel, we developed our PloidyNet WebPortal (Y1) as an interactive tool for the students and PIs to communicate among each other and with our project manager (WP1-management). All students have now started their individual research projects and quite a few have already spent time in labs other than their primary hosts’ as part of their secondments or industrial rotation. Furthermore, we organized focused courses on bioinformatics, high throughput screening, mouse modeling, microscopy, presentation skills and project planning, in addition to the courses that students took at their primary hosting institute (WP2-Training). Importantly, we also finished part I of the PloidyNet documentary on schedule in which students explain PloidyNet and their individual projects in laymen’s language, one of our Network-wide outreach activities (WP3-Outreach) as described in our grant agreement.

In addition to the Network general aims, we also defined a number of scientific aims to be addressed by our students, defined in Work Packages 4-8.
WP4 Molecular Causes of Aneuploidy: In this work package, students work together to better understand the molecular processed that lead to aneuploidy. To this aim, they are developing human artificial chromosomes (HACs) that, when engineered into human cell lines, can be used to ‘fish’ for and quantify proteins that accumulate at (aneuploid) kinetochores. In addition, students have developed mass spectrometry protocols to quantify expression of proteins important for signaling aneuploidy in various aneuploid cell lines that are now being employed to further characterize how the chromosome segregation machinery copes with abnormal chromosome numbers.
WP5 Physiological consequences of Aneuploidy: In this WP we study how cells respond to an aneuploid state. For this, students performed an RNA interference screen to identify several genes that signal abnormal karyotypes, for which candidate genes are currently being validated, and another screen to identify kinases that provoke apoptosis in response to CIN. In addition, we developed a biosensor for p53 activity (a key component in signaling aneuploidy) by endogenously tagging p53-protein. When validated, this tool will be translated into a mouse model (WP8-biosensors). Finally, we are characterizing primary non-small cell lung cancer (NSCLC) tumor samples and tumor-derived induced pluripotent stem cells to in depth analyze aneuploid tumor genomes using next generation sequencing, which is yielding a number of potentially interesting candidate genes involved in aneuploid tumor survival. The first data from this were published August 2015 in Cancer Discovery (Cancer Discov. 2015 Aug; 5(8): 821-31).
WP6 The role of Aneuploidy in Cancer Development: The aim of WP6 is to better understand how aneuploidy leads to cancer. To this aim, students make use of mouse models in which chromosomal instability is provoked by various means in the mammary gland. So far, the mouse models have been crossed to get the correct genotypes to develop aneuploid mammary tumors. In addition, students employ the recently developed protocols to culture tumors in a 3D setting in a dish (organoid culture) using colon carcinoma and mouse mammary tumor cells as a model system. Tumors and tumor cultures are and will be further characterized for karyotype heterogeneity using single cell sequencing and RNA sequencing.
WP7 Targeting Chromosome Segregation in Cancer Therapy: The aim of this work package is to train students how to drug mitotic pathways to best achieve efficient tumor cell death in vitro and in mice. As this work package depends on the success of new mitotic inhibitors being developed (e.g. based on the results on the RNA interference screen under WP5), students involved in WP7 are currently participating in other scientific WPs until these new inhibitors will have been uncovered. The first step in this WP has been the further characterization of an Mps1 inhibitor revealing that cells can actually cope much better with the inhibitor-induced CIN than previously anticipated, raising several new interesting questions.
WP8 Assay Development: Students participating in this WP are developing a number of new assays that will facilitate quantifying aneuploidy in vitro as well as in vivo. One tool through which PloidyNet research has progressed tremendously over the last year is single cell sequencing, by which we can now reliably assess full karyotypes of single non-dividing cells. Furthermore, we have engineered constructs and mouse ES cell lines to generate recombinant mice that express combinations of fluorescent mitotic markers (H2B-Centrin3-Tubulin or H2B-CenpB-Tubulin) in a conditional fashion, which will allow us to label (and lineage trace) cells in cell lineages of choice by intravital imaging. Finally, students are developing a flow cytometry-based assay to detect aneuploidy using cell surface markers. For this, we have are now validating ~70 candidates epitopes that appear to be differentially expressed in aneuploid cells, with a current focus on components of the integrin superfamily that appear to be deregulated as a consequence of aneuploidy.

In sum, PloidyNet has had a very successful start: management has successfully been implemented and training has started as scheduled and is already yielding very promising preliminary data. The E(S)Rs are presenting their work at (inter)national meetings, contributing to various outreach events and benefitting from the multidisciplinary nature of PloidyNet evidenced by their high mobility between PloidyNet labs even within the first year of their training.

Coordinator: Rene Medema
Director Netherlands Cancer Institute, Amsterdam, the Netherlands (http://www.nki.nl)
Web Portal: http://aneuploidy.nl

Contact

Henri Van Luenen, (director of operations NKI)
Tel.: +3120 5122097
E-mail

Subjects

Life Sciences
Record Number: 193201 / Last updated on: 2017-01-16
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