Biochemical networks maintain cellular homeostasis by processing information from the intra- and extra-cellular environments and coordinating appropriate responses to stimuli and stressors. During carcinogenesis, cells accrue genetic alterations that rewire signalling pathways, eventually leading to the partial loss of homeostatic control and cancer- related phenotypes. Cancer is a very heterogeneous disease with genetic alterations that exhibit vast variability within a tumour, between tumours of different tissues, and patients. Despite the enormous advances the community has achieved in understanding the molecular causes of cancer, the mechanisms of pathogenicity of specific mutations are often unclear, severely limiting the efficacy of the therapeutic intervention and disease management. Moreover, we are recognizing that driver mutations once considered sufficient to induce initiation and promotion of cancer are more frequent than originally expected also in healthy tissues. In addition to genetic variability, cell-to-cell variability of non-genetic origin is increasingly recognized – yet poorly understood – as a fundamental force fuelling carcinogenesis and resistance to therapies. We therefore develop biochemical imaging techniques to investigate aspects of cancer heterogeneity that are currently challenging – if not impossible – to study. KRAS is a driver gene significantly involved in lung, colorectal and pancreatic carcinogenesis. Various types of KRAS mutations respond differently to therapy, and some have been linked to the emergence of resistance. However, the mutation-specific mechanisms leading to these phenotypes is not well understood. We used KRAS as a clinically relevant model to study the effects of mutations at codon G12 on signalling dynamics and phenotypic changes, including cell-to-cell variability.

In this thesis, first I introduce the concepts of evolution-driven oncogenesis, genetic and non-genetic heterogeneity and signal transduction pathways. In Chapter 2, I describe the characterisation of KRAS mutation-specific MAPK signalling. To do this, I generated an isogenic panel of cells stably expressing a FRET sensor and developed an experimental pipeline including automated biochemical imaging, microfluidic-based stimulation, and custom image analysis. Using these tools, I have identified differ- ential signalling dynamics and heterogeneity in response to epidermal growth factor (EGF). Taken together with other results, we hypothesise the presence of a weakened negative feedback loop in cells with a G12D mutation in KRAS. In Chapter 3, I report on methodological developments aimed at drastically improving our capability to characterise biochemical networks in single living cells. I have optimized novel pairs of fluorescent proteins dedicated to biochemical multiplexing and capable of simultaneously monitoring three biochemical reactions. Moreover, I developed an ex- pandable software pipeline that yields single-cell ERK signalling data and several types of analyses from microscopy FRET imaging. Finally, I further develop optogenetic systems designed to dynamically and optically activate KRAS in single cells.

In conclusion, I show the effects of KRAS mutations on signalling dynamics and cell- to-cell variability, a first step towards a deeper understanding of how genetic and non-genetic heterogeneity may cooperate to make of cancer the terrible disease we know. At the same time, I have improved methodologies designed for the novel study of biochemical networks, laying down the foundations of system-level investigation in single living cells.