Differential Regulation of Growth-Promoting Signalling Pathways by E-Cadherin

Background: Despite the well-documented association between loss of E-cadherin and carcinogenesis, as well as the link between restoration of its expression and suppression of proliferation in carcinoma cells, the ability of E-cadherin to modulate growth-promoting cell signalling in normal epithelial cells is less well understood and frequently contradictory. The potential for E-cadherin to co-ordinate different proliferation-associated signalling pathways has yet to be fully explored. Methodology/Principal Findings: Using a normal human urothelial (NHU) cell culture system and following a calcium-switch approach, we demonstrate that the stability of NHU cell-cell contacts differentially regulates the Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-Regulated Kinase (ERK) and Phosphatidylinositol 3-Kinase (PI3-K)/AKT pathways. We show that stable cell contacts down-modulate the EGFR/ERK pathway, whilst inducing PI3-K/AKT activity, which transiently enhances cell growth at low density. Functional inactivation of E-cadherin interferes with the capacity of NHU cells to form stable calcium-mediated contacts, attenuates E-cadherin-mediated PI3-K/AKT induction and enhances NHU cell proliferation by allowing de-repression of the EGFR/ERK pathway and constitutive activation of beta-catenin-TCF signalling. Conclusions/Significance: Our findings provide evidence that E-cadherin can differentially and concurrently regulate specific growth-related signalling pathways in a context-specific fashion, with direct, functional consequences for cell proliferation and population growth. Our observations not only reveal a novel, complex role for E-cadherin in normal epithelial cell homeostasis and tissue regeneration, but also provide the basis for a more complete understanding of the consequences of E-cadherin loss on malignant transformation

Amphiregulin (areg) And Epidermal Growth Factor (egf): Disparate In Egfr Signaling And Trafficking

We have previously shown that SUM-149 human breast cancer cells require an AREG/EGFR autocrine loop for cell proliferation. We also demonstrated that AREG can increase EGFR stability and promote EGFR localization to the plasma membrane. In the presented dissertation we successfully knocked-down AREG expression in SUM-149 cells by lenti-viral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the cell surface. Soft agar assays demonstrated that AREG knock-down cells retain anchorage-independent growth capacity. Additionally mammosphere forming assays and Adefluor staining analysis showed that knock-down of AREG expression did not affect the expression of stem cell 158 phenotypes. However, following AREG knock-down, SUM-149 cells demonstrated a dramatic decrease in their ability to invade a Matrigel matrix. Consistent with this observation, microarray analysis comparing cells infected with a non-silencing vector to the AREG knock-down cells, identified genes associated with the invasive phenotype such as RHOB and DKK1, and networks associated with cell motility such as integrinlinked kinase signaling, and focal adhesion kinase signaling. AREG was also found to modulate WNT and Notch signaling in SUM-149 cells. In an additional microarray study, changes in gene expression were analyzed from cDNA transcribed from RNA isolated from MCF10A cells growing in the presence of AREG or EGF and after 24 hours withdrawl of the respective ligand. Genes regulating WNT signaling, but not NOTCH signaling, were altered in the MCF10A cells. Thus, the pathway that AREG/EGFR signaling effects is contextually dependent on the cell type that it is functioning in. We conclude that AREG functions in regulating the invasive phenotype, and we propose that this regulation may be through altered signaling that occurs when AREG activates plasma membrane localized EGFR

Stem cell plasticity and dormancy in the development of cancer therapy resistance

Cancer treatment with either standard chemotherapy or targeted agents often results in the emergence of drug-refractory cell populations, ultimately leading to therapy failure. The biological features of drug resistant cells are largely overlapping with those of cancer stem cells and include heterogeneity, plasticity, self-renewal ability, and tumor-initiating capacity. Moreover, drug resistance is usually characterized by a suppression of proliferation that can manifest as quiescence, dormancy, senescence, or proliferative slowdown. Alterations in key cellular pathways such as autophagy, unfolded protein response or redox signaling, as well as metabolic adaptations also contribute to the establishment of drug resistance, thus representing attractive therapeutic targets. Moreover, a complex interplay of drug resistant cells with the micro/macroenvironment and with the immune system plays a key role in dictating and maintaining the resistant phenotype. Recent studies have challenged traditional views of cancer drug resistance providing innovative perspectives, establishing new connections between drug resistant cells and their environment and indicating unexpected therapeutic strategies. In this review we discuss recent advancements in understanding the mechanisms underlying drug resistance and we report novel targeting agents able to overcome the drug resistant status, with particular focus on strategies directed against dormant cells. Research on drug resistant cancer cells will take us one step forward toward the development of novel treatment approaches and the improvement of relapse-free survival in solid and hematological cancer patients

A diversity-aware computational framework for systems biology

L'abstract è presente nell'allegato / the abstract is in the attachmen

Data-driven modelling of biological multi-scale processes

Biological processes involve a variety of spatial and temporal scales. A holistic understanding of many biological processes therefore requires multi-scale models which capture the relevant properties on all these scales. In this manuscript we review mathematical modelling approaches used to describe the individual spatial scales and how they are integrated into holistic models. We discuss the relation between spatial and temporal scales and the implication of that on multi-scale modelling. Based upon this overview over state-of-the-art modelling approaches, we formulate key challenges in mathematical and computational modelling of biological multi-scale and multi-physics processes. In particular, we considered the availability of analysis tools for multi-scale models and model-based multi-scale data integration. We provide a compact review of methods for model-based data integration and model-based hypothesis testing. Furthermore, novel approaches and recent trends are discussed, including computation time reduction using reduced order and surrogate models, which contribute to the solution of inference problems. We conclude the manuscript by providing a few ideas for the development of tailored multi-scale inference methods.Comment: This manuscript will appear in the Journal of Coupled Systems and Multiscale Dynamics (American Scientific Publishers

Novel descriptive and model based statistical approaches in immunology and signal transduction

Biological systems are usually complex nonlinear systems of which we only have a limited understanding. Here we show three different aspects of investigating such systems. We present a method to extract detailed knowledge from typical biological trajectory data, which have randomness as a main characteristic. The migration of immune cells, such as leukocytes, are a key example of our study. The application of our methodology leads to the discovery of novel random walk behaviour of leukocyte migration. Furthermore we use the gathered knowledge to construct the under- lying mathematical model that captures the behaviour of leukocytes, or more precisely macrophages and neutrophils, under acute injury. Any model of a biological system has little predictive power if it is not compared to collected data. We present a pipeline of how complex spatio- temporal trajectory data can be used to calibrate our model of leukocyte migration. The pipeline employs approximate methods in a Bayesian framework. Using the same approach we are able to learn additional information about the underlying signalling network, which is not directly apparent in the cell migration data. While these two methods can be seen as data processing and analysis, we show in the last part of this work how to assess the information content of experiments. The choice of an experiment with the highest information content out of a set of possible experiments leads us to the problem of optimal experimental design. We develop and implement an algorithm for simulation based Bayesian experimental design in order to learn parameters of a given model. We validate our algorithm with the help of toy examples and apply it to examples in immunology (Hes1 transcription regulation) and signal transduction (growth factor induced MAPK pathway)

Osimani B., Poellinger R. (2020) A Protocol for Model Validation and Causal Inference from Computer Simulation. In: Bertolaso M., Sterpetti F. (eds) A Critical Reflection on Automated Science. Human Perspectives in Health Sciences and Technology, vol 1. Springer, Cham. https://doi.org/10.1007/978-3-030-25001-0_9

The philosophical literature on modelling is increasingly vast, however clear formal analyses of computational modelling in systems biology are still lacking. We present a general, theoretical scheme which (i) visualizes the development and repeated refinement of a computer simulation, (ii) explicates the relation between different key concepts in modelling and simulation, and (iii) facilitates tracing the epistemological dynamics of model validation. To illustrate and motivate our conceptual scheme, we analyse a case study, the discovery of the functional properties of a specific protein, E-cadherin, which seems to have a key role in metastatic processes by way of influencing cell growth and proliferation signalling. To this end we distinguish two types of causal claims inferred from a computer simulation: (i) causal claims as plain combinations of basic rules (capturing the causal interplay of atomic behaviour) and (ii) causal claims on the level of emergent phenomena (tracing population dynamics). In formulating a protocol for model validation and causal inference, we show how, although such macro-level phenomena cannot be subjected to direct causal tests qua intervention (as, e.g., formulated in interventionist causal theories), they possibly suggest further manipulation tests at the basic micro-level. We thereby elucidate the micro-macro-level interaction in systems biology

RAB5A IN THE CONTROL OF MAMMARY EPITHELIAL MORPHOGENESIS AND MOTILITY

Endocytosis has been originally considered as a fundamental mechanism involved in the internalization of nutrients and membrane-bound molecules into the cell. However, recent studies demonstrate that endocytosis is a more complex network that regulates the delivery of specific time- and space-resolved signals to the cell. The small GTPase RAB5A, a master regulator of endocytosis, is emerging as a key player in tumor progression and metastatic dissemination. Consistently, the expression of RAB5A has been shown to promote a tumor mesenchymal invasive program and to correlate with the metastatic potential of different tumors. In Drosophila, however, loss-of-function mutant of RAB5 transforms imaginal disc epithelia into highly proliferative tissues, pointing to a tumor suppressor function of RAB5. To dissect the complex role of this GTPase in tumor development, we investigated the impact of RAB5A deregulation on MCF-10A, an immortalized non-transformed mammary epithelial cell line that forms hollow acinar-like spheroids recapitulating the morphogenesis of the human mammary gland, when cultured on 3D reconstituted basement membrane. We generated stable and inducible MCF-10A cells expressing either RAB5A WT or its dominant negative form (RAB5AS34N). We found that the expression of RAB5AS34N is sufficient to sustain MCF-10A cells proliferation in the absence of EGF. We identified Amphiregulin, a known EGFR ligand, as the secreted diffusible factor responsible for EGF-independent proliferation. Conversely, the expression of RAB5A WT delayed cell 15 cycle progression of cells grown in 2D, albeit it promoted the formation of hyper- proliferative acini when these cells were grown in 3D, without affecting acini morphological architecture or polarity establishment. Thus, RAB5A may either be implicated in growth factor independent growth, or may promote proliferation in 3D. Both clinical data and in vitro studies showed that RAB5A is required for invasion and metastasis formation, suggesting its involvement in tumor progression. In particular, the overexpression of RAB5A has been shown to be predictive of aggressive behavior and metastatic ability in human breast cancer. To further explore this function, we investigated the role of RAB5A in MCF-10A cells motility. We demonstrated that RAB5A expression does not affect single cell migration, but specifically enhances collective locomotion, the typical motility mode frequently observed in invasive cancer of epithelial origin, such as breast carcinoma. Indeed, RAB5A expression promotes increased speed and coordination of the epithelial cell sheet motility, related both to the increase in the area and persistence of migratory protrusions in cells at the leading front, and possibly to a tightening of cell-to- cell contacts, which improves cohesiveness of the migrating epithelial sheet. In conclusion, our data suggest a temporally distinct dual role of RAB5A in tumor development. On one hand, RAB5A may exploit a tumor suppressor function controlling epithelial tissue morphogenesis and homeostasis, which impairment may induce tumor initiation. On the other hand, RAB5A promotes not only a mesenchymal program of individual cell invasion, as previously shown by our group, but also it enhances coordinated collective epithelial migration, thus promoting cancer progression and dissemination