ErbB Ligands in Angiogenesis

The formation of new blood vessels, i.e. angiogenesis, is an important phenomenon during normal development and wound repair, as well as during various pathological processes, such as tumor growth and metastasis. Specific growth factors regulate angiogenesis by modulating the different cellular functions of endothelial cells (EC), and periendothelial cells, such as pericytes (PC) and smooth muscle cells (SMC), which interact with ECs in a paracrine manner. ErbB receptors form a subgroup of transmembrane receptor tyrosine kinases that interact with growth factors of the epidermal growth factor (EGF) family. ErbB receptors regulate behaviour of a variety of normal as well as tumor cell types. Cancer drugs that target epidermal growth factor receptor (EGFR, ErbB1) or ErbB2 receptor have been approved for clinical use. It has been speculated that part of the antitumor activity of ErbB inhibitor compounds result from an antiangiogenic mechanism. The results presented here indicate a role for endothelial-derived EGF-like growth factors heparin binding EGF-like growth factor (HB-EGF) and neuregulin-1 (NRG-1) in the paracrine regulation of angiogenesis. HB-EGF, EGFR and ErbB2 are shown to mediate interaction between ECs and SMCs in vitro, and gefitinib, an inhibitor of EGFR kinase activity, suppresses recruitment of PCs/SMCs in vivo. NRG-1 is shown to regulate EC functions in vitro and angiogenesis in vivo by indirect mechanisms involving vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2). Furthermore, EGFR activity is demonstrated to regulate recruitment of bone marrow-derived perivascular cells during tumor neovascularization in vivo. These results indicate that ErbB signaling is involved in the cellular processes of new blood vessel formation. This study gives new information about the role of ErbB ligands and receptors in angiogenesis and vasculogenesis and about the mechanisms by which ErbB inhibitor drugs such as gefitinib affect tumor growth.

Role of ErbB2 and ErbB4 in Cancer Growth, Prognosis and as Targets for Immunotherapy

ErbB receptors (EGFR, ErbB2, ErbB3 and ErbB4) are growth factor receptors that regulate signals of cell differentiation, proliferation, migration and survival. Inappropriate activation of these receptors is associated with the development and severity of many cancers and has prognostic and predictive value in cancer therapy. Drugs, such as therapeutic antibodies, targeted against EGFR and ErbB2, are currently used in therapy of breast, colorectal and head and neck cancers. The role of ErbB4 in tumorigenesis has remained relatively poorly understood. Alternative splicing produces four different isoforms of one ErbB4 gene. These isoforms (JM-a, JM-b, CYT-1 and CYT-2) are functionally dissimilar and proposed to have different roles in carcinogenesis. The juxtamembrane form JM-a undergoes regulated intramembrane proteolysis producing a soluble receptor ectodomain and an intracellular domain that translocates into the nucleus and regulates transcription. Nuclear signaling via JM-a isoform stimulates cancer cell proliferation. This study aimed to develop antibodies targeting the proposed oncogenic ErbB4 JM-a isoform that show potential in inhibiting ErbB4 dependent tumorigenesis. Also, the clinical relevance of ErbB4 shedding in cancer was studied. The currently used monoclonal antibody trastuzumab, targeting ErbB2, has shown efficacy in breast cancer therapy. In this study novel tissues with ErbB2 amplification and trastuzumab sensitivity were analyzed. The results of this study indicated that a subpopulation of breast cancer patients demonstrate increased shedding and cleavage of ErbB4. A JM-a isoform-specific antibody that inhibited ErbB4 shedding and consequent activation of ErbB4 had anti-tumor activity both in vitro and in vivo. Thus, ErbB4 shedding associates with tumor growth and specific targeting of the cleavable JM-a isoform could be considered as a strategy for developing novel ErbB-based cancer drugs. In addition, it was demonstrated that ErbB2 amplification is common in intestinal type gastric cancers with poor clinical outcome. Trastuzumab inhibited growth of gastric and breast cancer cells with equal efficacy. Thus, ErbB2 may be a useful target in gastric cancer.

Regulation of cell growth, death, and polarization by ERBB4

Regulation of cell growth, death, and polarization by ERBB4 ErbB4 is a member of the epidermal growth factor receptor (EGFR, ErbB) family. The other members are EGFR, ErbB2 and ErbB3. ErbB receptors are important regulators for example in cardiovascular, neural and breast development but control key cellular functions also in many adult tissues. Abnormal ErbB signaling has been shown to be involved in various illnesses such as cancers and heart diseases. Among the ErbBs, ErbB4 has been shown to have unique signaling characteristics. ErbB4 exists in four alternatively spliced isoforms that are expressed in a tissue-specific manner. Two of the isoforms can be cleaved by membrane proteases, resulting in release of soluble intracellular domains (ICD). Once released into the cytosol, the ICD is capable of translocating into the nucleus and participating in regulation of transcription. The functional differences and the tissue-specific expression patterns suggest isoformspecific roles for ErbB4 isoforms. However, the abilities of ErbB4 isoforms to differently regulate cellular functions were discovered only recently and are not well understood. This study aimed to determine the expression patterns of ErbB4 in normal and diseased tissue, and to define whether the cleavable and non-cleavable isoforms could regulate different target genes and therefore, cellular functions. In this study, a comprehensive ErbB4 expression pattern in several normal tissues, various cancers and non-neoplastic diseases was determined. In addition, the data demonstrated that the cleavable and non-cleavable ErbB4 isoforms could regulate different cellular functions and target genes. Finally, this study defined the cellular responses regulated by ErbB4 during kidney development.

Novel Functions of ErbB4 and NRG-1 in Development and Cancer

Cells communicate, or signal, with each other constantly to ensure proper functioning of tissues and organs. Cell signaling is often performed by interplay of receptors and ligands that bind these receptors. ErbB receptors (epidermal growth factor receptors, EGFR, HER) bind extracellular growth factors and transduce these signals inside of cells. ErbB dysfunction promotes carcinogenesis, and also results in numerous defects during normal development. This study focused on the functions of one member of the ErbB receptor family, ErbB4, and growth factor, neuregulin-1 (NRG-1), that can bind and activate ErbB4. This study aimed to find novel functions of ErbB4 and NRG-1. Hypoxia, or deficiency of oxygen, is common in cancer and ischemic conditions. One of the key findings of the work was the identification and characterization of a cross-talk between ErbB4 and Hypoxia-inducible factor 1α (HIF-1α), the central mediator of hypoxia signaling. ErbB4 activation by NRG-1 was found to increase HIF-1α activity. Interestingly, this regulation occurred in reciprocal manner as HIF-1α was also able to increase protein levels of NRG-1 and ErbB4. Moreover, expression of NRG-1 and ErbB4 was associated with HIF activity in vivo in human clinical samples and in mice. Reduction of functional ErbB4 in developing zebrafish embryos resulted in defects in development of the skeletal muscles. To study ErbB4 functions in pathological situation in humans, clinical samples of serous ovarian carcinoma were analyzed using tissue microarrays and real-time RT-PCR. A specific isoform of ErbB4, CYT-1, was associated with poor survival in serous ovarian cancer and increased anchorage independent growth of ovarian cancer cells in vitro. These observations demonstrate that ErbB4 and NRG-1 are essential regulators of cellular response to hypoxia, of development, and of ovarian carcinogenesis.

ERBB4 mutations in cancer and amyotrophic lateral sclerosis

ErbB receptor tyrosine kinases, epidermal growth factor receptor (EGFR, also known as ErbB1), ErbB2 (HER2 or NEU), ErbB3 (HER3), and ErbB4 (HER4), transduce signals borne by extracellular ligands into central cellular responses such as proliferation, survival, differentiation, and apoptosis. Mutations in ERBB genes are frequently detected in human malignant diseases of epithelial and neural origin, making ErbB receptors important drug targets. Targeting EGFR and ErbB2 has been successful in eg. lung and breast cancer, respectively, and mutations in these genes can be used to select patients that are responsive to the targeted treatment. Although somatic ERBB4 mutations have been found in many high-incidence cancers such as melanoma, lung cancer, and colorectal cancer and germ-line ERBB4 mutations have been linked to neuronal disorders and cancer, ErbB4 has generally been neglected as a potential drug target. Thus, the consequences of ERBB4 mutations on ErbB4 biology are largely unknown. This thesis aimed to elucidate the functional consequences and assess the clinical significance of somatic and germ-line ERBB4 mutations in the context of cancer and amyotrophic lateral sclerosis. The results of this study indicated that cancer-associated ERBB4 mutations can promote aberrant ErbB4 function by activating the receptor or inducing qualitative changes in ErbB4 signaling. ERBB4 mutations increased survival or decreased differentiation in vitro, suggesting that ERBB4 mutations can be oncogenic. Importantly, the potentially oncogenic mutations were located in various subdomains in ErbB4, possibly providing explanation for the characteristic scattered pattern of mutations in ERBB4. This study also demonstrated that hereditary variation in ERBB4 gene can have a significant effect on the prognosis of breast cancer. In addition, it was shown that hereditary or de novo germ-line ERBB4 mutations that predispose to amyotrophic lateral sclerosis inhibit ErbB4 activity. Together, these results suggest that ErbB4 should be considered as a novel drug target in cancer and amyotrophic lateral sclerosis.

Quantitative refinement of reaction-based biomodels

In the field of molecular biology, scientists adopted for decades a reductionist perspective in their inquiries, being predominantly concerned with the intricate mechanistic details of subcellular regulatory systems. However, integrative thinking was still applied at a smaller scale in molecular biology to understand the underlying processes of cellular behaviour for at least half a century. It was not until the genomic revolution at the end of the previous century that we required model building to account for systemic properties of cellular activity. Our system-level understanding of cellular function is to this day hindered by drastic limitations in our capability of predicting cellular behaviour to reflect system dynamics and system structures. To this end, systems biology aims for a system-level understanding of functional intraand inter-cellular activity. Modern biology brings about a high volume of data, whose comprehension we cannot even aim for in the absence of computational support. Computational modelling, hence, bridges modern biology to computer science, enabling a number of assets, which prove to be invaluable in the analysis of complex biological systems, such as: a rigorous characterization of the system structure, simulation techniques, perturbations analysis, etc. Computational biomodels augmented in size considerably in the past years, major contributions being made towards the simulation and analysis of large-scale models, starting with signalling pathways and culminating with whole-cell models, tissue-level models, organ models and full-scale patient models. The simulation and analysis of models of such complexity very often requires, in fact, the integration of various sub-models, entwined at different levels of resolution and whose organization spans over several levels of hierarchy. This thesis revolves around the concept of quantitative model refinement in relation to the process of model building in computational systems biology. The thesis proposes a sound computational framework for the stepwise augmentation of a biomodel. One starts with an abstract, high-level representation of a biological phenomenon, which is materialised into an initial model that is validated against a set of existing data. Consequently, the model is refined to include more details regarding its species and/or reactions. The framework is employed in the development of two models, one for the heat shock response in eukaryotes and the second for the ErbB signalling pathway. The thesis spans over several formalisms used in computational systems biology, inherently quantitative: reaction-network models, rule-based models and Petri net models, as well as a recent formalism intrinsically qualitative: reaction systems. The choice of modelling formalism is, however, determined by the nature of the question the modeler aims to answer. Quantitative model refinement turns out to be not only essential in the model development cycle, but also beneficial for the compilation of large-scale models, whose development requires the integration of several sub-models across various levels of resolution and underlying formal representations.