Research

The interest of the lab is to understand and control molecular mechanisms that regulate cell fate. The focus is the evolutionarily conserved Notch pathway, a key regulator of stem cells in most tissues and essential for development. Deregulation of the pathway is the cause of several diseases, most notably cardiovascular diseases, and cancer, where it is linked to aggressive, therapy-resistant cancers.

Key aims of the research group are:

Current projects:

Fine tuning of Notch signaling can be achieved by crosstalk with other signaling pathways and by posttranslational modifications (PTMs). Notch signaling is regulated by PTMs that influence nuclear translocation, target gene expression, and half-life of Notch intracellular domain (NICD).

We are currently focused on bringing into light how the activity of the Notch signaling pathway is mechanistically regulated at a posttranslational level by small ubiquitin-like modifiers (SUMO). The concepts we are addressing are being approached by using both in vitro and in vivo tools, which have revealed various aspects in how SUMO alters the fate of an already activated Notch pathway in a context-dependent manner. Dynamic expression of Notch target genes is crucial for development and cellular responses to changes in the physiological environment.

We have identified site-specific phosphorylation events which are important for Notch receptor trafficking and activity during stem cell differentiation and are currently elucidating how receptor isoform specific phosphorylation affects oncogenic Notch function. We have also identified novel regulators of Jagged, one of the ligands of Notch, trafficking by siRNA based high through put cell arrays and is currently evaluating the functionality of these regulators.

Since only few PTMs of Notch have been identified to date, elucidating how PTMs alters Notch activity and its target gene expression provides a novel regulatory aspect of the of the pathway, which may be amendable to therapeutic intervention.

In our research we glimpse behind the veil into the unknowns of Notch signaling and investigate several novel aspects and peculiarities relating to Notch deregulation in cancer. We also take great interest in understanding how differential regulation of Notch translates into distinct physiological effects and how our findings can be used to answer clinically relevant questions.

We have shown that Notch signaling is able to reprogram the metabolism in breast cancer (Landor et al. 2011). Increase of Notch levels promotes tumor growth in vivo, as well as in vitro spheroid formation and in vivo tumor formation from a low number of xenografted cancer cells. The latter observations are typical characteristics of cancer stem cells (CSCs), a specialized type of cancer cells associated with treatment resistance and the capability of clonal expansion and tumor recapitulation. Notch activation can allow estrogen-dependent breast cancer cells to form tumors in vivo in estrogen-lacking (gonadectomized) hosts, demonstrating that Notch can convey estrogen-independency and, furthermore, hormone therapy-resistance, in breast cancer (Mamaeva et al. 2016).

Current activities are focused on investigating the interplay between Notch and oncogenic Pim kinases in both breast and prostate cancer. We are further focusing on the role of Notch in the interplay between different cellpopulations in the tumor microenvironment and how the biophysical tumor environment influences Notch signaling and metastatic behavior.

The Notch signaling pathway is a crucial regulator of multiple aspects of vascular development, including angiogenesis, vasculogenesis, differentiation and vascular patterning and remodeling by vascular smooth muscle cells. Blood vessels are constantly subjected to hemodynamic mechanical cues, such as stretch, shear stress and pressure. Deregulation or mutations in the Notch pathway is linked to disorganized and malfunctioning vasculature and to a number of cardiovascular disorders associated with biomechanical imbalance. These include disorders caused by narrowing of the coronary arteries due to atherosclerosis and coronary heart diseases (CHD), pulmonary arterial hypertension (PAH), the adult-onset genetic stroke syndrome CADASIL and the Alagille syndrome. Further Notch critically regulates tumor angiogenesis. Our current research activities are focused on elucidating mechanisms regulating Notch ligand and receptor function in the vasculature. We are especially interested in the crosstalk between different cells in the tumor environment and how the hemodynamic microenvironment influences Notch signaling in vascular development and remodeling.

Given the complexity and dual-faceted nature of Notch signalling in oncogenesis, where Notch appears to fuel malignant progression in certain types of cancer (e.g., lymphoproliferative malignancies and breast cancer), while inhibiting growth of others (e.g., head and neck carcinomas, selected neuroendocrine tumours), it comes as no surprise that the majority of attempts of pharmacological modulation of Notch signalling by systemic administration of Notch inhibitors and activators have not produced clinically-relevant results. Besides that, poor pharmacokinetic and issues with tolerability further hinder the successful clinical translation of Notch activity modulators.

With all the above bottlenecks in mind, our lab is trying to develop novel modes of Notch activity modulation at the interphase of tumor biology and nanotechnology, by harnessing engineered and highly biocompatible silica nanoparticles as vehicles for selective delivery of Notch modulators to different cancers. A key collaborator is Professor Jessica Rosenholm, Åbo Akademi University who has extensive experience in nanoparticle synthesis and functionalization for biomedical application.

Our expertise in cancer biology coupled with flexible formulation approaches allow for insightful design of nanocarriers, tailored for particular malignancies. Successful stories from our lab include in vivo routing of folate-decorated and Notch inhibitors-loaded silica nanocarriers to breast cancer cells with aberrant Notch up-regulation and selective modulation of Notch signalling in breast cancer stem cells with sugar functionalized nanocarriers. Another project on the way involves targeting of custom-made silica particles to digestive neuroendocrine malignancies by virtue of G protein – coupled receptors for Notch activation, thus restoring tumor suppressor function of Notch in this type of cancer.

We believe that the exciting results obtained with nanocarriers in our lab so far will ultimately help to bridge this delivery platform to the clinics, opening new avenues for precise and directional modulation of oncogenes and tumor-suppressor genes in selected malignancies. We are currently extending the activities to regenerative medicine by designing biofunctional scaffolds for stem cell based tissue regeneration through spatial and temporal control of Notch activity.

Angiogenesis is a process where new blood vessels emerge from existing vessels when there is need for new vasculature to provide nutrients and eliminating waste or during tissue growth or repair. The process is triggered by the low oxygen levels in surrounding tissue. The Notch signaling pathway plays a pivotal role the regulation of angiogenesis, as it is believed to act as determining factor of cell fate of the endothelial cells residing in the blood vessels. Under hypoxia, Dll4-Notch1 signaling is responsible for the differentiation of endothelial cells into migratory tip cells or proliferating stalk cells.

By manipulation of the presentation of the responsible Notch signaling ligands to the endothelial cells with microfabrication techniques in vitro, we aim to gain control over the angiogenic process and thereby manipulate the formation of (neo) micro vessels. Secondly, we hope to gain more insight over the amount of control over angiogenesis that is possible and apply this into (biomaterial) solutions for cardiovascular tissue engineering.

Notch signalling is a direct cell-cell communication pathway that plays a key role in cardiovascular development. Specific removal of different Notch receptors and ligands has shown that they are essential for the formation of a mature vasculature. Mutations in Notch related proteins can lead to different cardiovascular diseases like CADASIL, Alagille syndrome or calcific aortic valve disease.

Our group is interested in the role of Notch signalling in vascular organization with a specific focus on mechanobiology. To understand this relationship, we study endothelial cells exposed to shear stress, smooth muscle cells exposed stretch or the interaction between these cell types in response to mechanics. Furthermore, we have found that vimentin, one of the components of the cytoskeleton and family of the intermediate filaments, is interrelated with Notch signalling. However, these studies are still inconclusive and do not give mechanistic insights.

The goal in this research is to investigate the role of hemodynamic loading on our previously identified link between vimentin and notch signaling in vascular cells. It is believed that studies are likely influenced by the experimental design, as in vitro studies are lacking the correct cellular organization and composition compared to the physiological tissue. Furthermore, poor control of the hemodynamic environment is an essential limitation of the in vivo studies compared to the in vitro studies. Therefore, we will combine different in vivo and in vitro approaches to answer our research question. Detailed understanding of the interplay between the mechanical influence of blood flow and vascular remodeling will identify new therapeutic targets and help predict therapeutic outcomes in vascular medicine and engineering.

Metastasis is preceded by invasion of cancer cells from the primary tumour through the extracellular matrix (ECM) into the surrounding tissue. ECM stiffness has been identified as an important factor regulating tumour invasiveness and a stiff ECM is heavily implicated as a promoter of breast cancer and its malignant progression. Deregulation of Notch signalling is in many ways linked to cancer and plays either an oncogenic or tumour suppressive role depending on the context. In relation to breast cancer, the receptor Notch1 and ligand Jagged1 promote epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells and their high expression is correlated with increased mortality of breast cancer patients. The purpose of the current study is to determine how ECM stiffness affects expression of Notch signalling components and Notch activity in different types of breast-derived cells and to study whether Notch signalling mediates or regulates the invasive and metastatic effect of a stiff ECM. Different types of established and novel 2D and 3D scaffolds will be used as ECM mimetics in the study.

Projects in Eindhoven:

Link

Cell-based cardiac regenerative therapies aim at restoring heart function and structure following cardiac disease, such as myocardial infarction. Human endogenous cardiomyocyte progenitor cells (CMPCs) are considered a valuable cell source since they are able to differentiate into cardiomyocytes and have shown to improve cardiac function when injected in the injured heart. To improve the effect of stem cell therapy, the injection of stem cells can be combined with a biomaterial, which can ameliorate cell retention, survival and differentiation. When injected in the injured myocardium, cells encounter a hostile environment, due to the altered mechanical properties of infarcted heart, and their mechanoresponse can dramatically influence the outcome of the treatment. In particular, the injected cells should be able to align with the local cardiomyocytes, in order to achieve a more homogeneous distribution of the electrical signal conduction and force generation, with an overall better therapeutic outcome.

The mechanism used by cells to sense and respond to biomechanical cues is called mechanotransduction, and is mainly regulated by cell-matrix adhesion proteins, i.e. integrins and integrin-linked proteins (e.g. vinculin, FAK, talin).

This project is focused on studying cardiac progenitor cell (CMPC) response to mechanical stimuli, with a focus on the development and activation of their mechanosensing apparatus and the key signaling mechanisms that regulate CMPC mechanotransduction.

Link

Heart valve disease remains a major health problem. Annually approximately 300.000 valve replacements are performed worldwide and this number is expected to be tripled in 2050. With the current valve replacements half of the patients will come back to the clinic with prosthesis related complications within 10-15 years. Especially for young patients this is a problem. In situ tissue engineering could provide an outcome by implanting a heart valve that uses the regenerative capacity of the human body. Over time the implant will degrade and a native heart valve will remain.

The native heart valve consists of three different layers with each a distinct function. One of the challenges in in situ tissue engineering is the development of this multilayered tissue. It is known from previous studies that Notch signaling plays a prominent role in vascular development.

In this project we would like to understand the relationship between Notch signaling, hemodynamic forces and cardiovascular tissue organization. With this knowledge we could modulate our scaffold in such a way that it can guide multilayered tissue formation via Notch signaling.

Link

The Notch cell-to-cell signalling pathway is a powerful cue to direct cell fate. Defects in various Notch proteins lead to a spectrum of cardiovascular diseases, ranging from valve calcification to infarcts of small arteries. All of these diseases are related to the cells taking a wrong turn when deciding on their fate.

Since the Notch pathway is such a powerful cue and highly dose-dependent, cells use multiple mechanisms of regulation to control the amount of active signalling. These mechanisms include transcription, translation, post-translational modifications, trafficking, endocytosis, recycling and degradation regulation.

To be able to use the Notch pathway to direct the fate of tissue-engineered heart valves, we will study the cross-talk of the Notch pathway with the hemodynamic forces that the implants will be exposed to in the heart valves. We work with shear and/or strain models to discover effects on Notch regulation, Notch activity, and ultimately cell fate.

This knowledge will be implemented by incorporating activators or inhibitors of Notch signalling in the scaffolds we use for tissue-engineering. With this we aim to direct the tissue generating cells to mimic healthy tissue as closely as possible.