Metabolic regulation of proliferation and differentiation
Authors:   , Ludikhuize Marlies Corine 
Published: April 2023

Accumulating evidence shows that metabolism, cellular signaling and epigenetics are highly intertwined. Metabolic alterations, therefore, affect cellular behavior. This thesis focuses on deepening our understanding of metabolic regulation of proliferation and differentiation, two cellular processes that are involved in tissue homeostasis and (re)generation. In chapter 2, we provide a literature review of the metabolic transitions occurring during stem cell activation and differentiation in different tissue types. These metabolic transitions are tissue type specific and can be a requisite for stem cell proliferation and differentiation. We additionally describe that Forkhead box O transcription factors (FOXOs) coordinate stem cell proliferation and differentiation through various mechanisms, including regulating cell cycle and stem cell factors, scavenging of oxygen radicals, and coordination of metabolic processes. In chapter 3, we study to how FOXO transcription factors are involved in the function and differentiation of intestinal stem cells. We use mice and mouse small intestinal organoids as model systems. We show that FOXOs affect Notch signaling, a classic signaling pathway involved in intestinal stem cell differentiation. We observe that loss of FOXOs or Notch signaling induces differentiation towards secretory cells, such as Paneth cells or goblet cells. Additionally, we show that loss of both FOXOs and Notch signaling results in mitochondrial fragmentation, which is a required step for secretory cell differentiation. Loss of the balance between proliferation and differentiation can cause colorectal cancer. To generate sufficient building blocks to sustain rapid division, cancer cells reprogram their metabolism. Cancer cells generally show high rates of glycolysis and anabolic pathways. The enhanced nucleotide synthesis is targeted by the commonly used chemotherapy 5-fluorouracil (5-FU). This chemotherapy is often not effective and can cause severe side effects. Moreover, it is hard to predict which patients will benefit from 5-FU treatment. In chapters 4 and 5, we study the molecular mechanisms underlying 5-FU toxicity in healthy colon organoids and colorectal cancer organoids. In healthy colon organoids, 5-FU caused P53 activation through DNA damage and redox signaling. Active P53 induced a G1 arrest, which protected against DNA damage accumulation and cell death in healthy organoids. In p53-deficient organoids, 5-FU induced DNA damage accumulation and cell death in proliferating cells due to impaired cell cycle arrest. This cell death was independent of oxidative stress. Moreover, we find that targeting the Warburg effect in p53-deficient and KRASG12D glycolytic tumor organoids enhances 5-FU toxicity by further altering the nucleotide pool and, importantly, without affecting non-transformed WT cells. Thus, p53 emerges as an important factor in the 5-FU response, and targeting cancer metabolism in combination with replication stress-inducing chemotherapies emerges as a promising strategy for CRC treatment. In Chapter 6, we describe a protocol for the application of Seahorse XF technology to organoids. Applying this technique to colon (cancer) organoids enables for example studying the energy metabolism of specific cell types in the colon or of different tumor types. Taken together, this thesis brings us one-step closer to unravelling the complex metabolic processes and interactions regulating cellular behavior, driving cancer development and determining treatment sensitivity.