An abundant supply of amino acids is important for cancers to sustain their proliferative drive. Alongside their direct role as substrates for protein synthesis, they can have roles in energy generation, driving the synthesis of nucleosides and maintenance of cellular redox homoeostasis. As cancer cells exist within a complex and often nutrient-poor microenvironment, they sometimes exist as part of a metabolic community, forming relationships that can be both symbiotic and parasitic. Indeed, this is particularly evident in cancers that are auxotrophic for particular amino acids. This review discusses the stromal/cancer cell relationship, by using examples to illustrate a number of different ways in which cancer cells can rely on and contribute to their microenvironmentboth as a stable network and in response to therapy. In addition, it examines situations when amino acid synthesis is driven through metabolic coupling to other reactions, and synthesis is in excess of the cancer cell's proliferative demand. Finally, it highlights the understudied area of non-proteinogenic amino acids in cancer metabolism and their potential role.

Highlights d Proline synthesis through PYCR1 is increased in low-oxygen conditions d PYCR1 activity in hypoxia supports TCA cycle function through NADH oxidation d PYCR1 is required for maintenance of hypoxic tumor regions

Proline is a non-essential amino acid with key roles in protein structure/function and maintenance of cellular redox homeostasis. It is available from dietary sources, generated de novo within cells, and released from protein structures; a noteworthy source being collagen. Its catabolism within cells can generate ATP and reactive oxygen species (ROS). Recent findings suggest that proline biosynthesis and catabolism are essential processes in disease; not only due to the role in new protein synthesis as part of pathogenic processes but also due to the impact of proline metabolism on the wider metabolic network through its significant role in redox homeostasis. This is particularly clear in cancer proliferation and metastatic outgrowth. Nevertheless, the precise identity of the drivers of cellular proline catabolism and biosynthesis, and the overall cost of maintaining appropriate balance is not currently known. In this review, we explore the major drivers of proline availability and consumption at a local and systemic level with a focus on cancer. Unraveling the main factors influencing proline metabolism in normal physiology and disease will shed light on new effective treatment strategies.

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The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a Hypoxia-Inducible Factor (HIF) target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting β-cells in vivo. Here, we show that deletion of PHD3 specifically in β-cells (βPHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets is associated with impaired glucose-stimulated ATP/ADP rises, Ca 2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the β-cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.

22 23Character count (excluding abstract, references and figure legends). 37,922 24 25 2 ABSTRACT 26The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a hypoxia-27 inducible factor target that uses molecular oxygen to hydroxylate proline. While PHD3 has 28 been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little 29is known about effects of this highly conserved enzyme in insulin-secreting β-cells. Here, we 30show that deletion of PHD3 specifically in β-cells (βPHD3KO) is associated with impaired 31 glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, 32 βPHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate 33 and a shift away from glycolysis. However, βPHD3KO islets are able to maintain oxidative 34 phosphorylation and insulin secretion by increasing utilization of fatty acids to supply the 35 tricarboxylic acid cycle. This nutrient-sensing switch cannot be sustained and βPHD3KO islets 36 begin to show signs of failure in response to prolonged metabolic stress, including impaired 37 glucose-stimulated ATP/ADP rises, Ca 2+ fluxes and insulin secretion. Thus, PHD3 might be a 38 pivotal component of the β-cell glucose metabolism machinery by suppressing the use of fatty 39 acids as a primary fuel source, under obesogenic and insulin resistant states. 40 Keywords: beta cell, insulin, metabolic stress, hypoxia, prolyl hydroxylase domain proteins, 41 PHD3, Egln3. 42 43 SIGNIFICANCE STATEMENT 44

Hypoxia is a common feature of glioblastoma, and a known driver of therapy resistance in brain tumours. Understanding the metabolic adaptations to hypoxia is key to develop new effective treatments for patients. A recent screening study highlighted Pyrroline-5-carboxylate reductase-like (PYCRL) as one of the top three genes that allowed tumour survival in hypoxia. PYCRL is one of the three enzymes involved in proline biosynthesis along with the mitochondrial pyrroline-5-carboxylate reductase 1 and 2 (PYCR1/2). The latter use glutamine as the carbon source to fuel the pyrroline-5-carboxylate (P5C)-to-proline reaction, whereas the cytosolic PYCRL is known to use ornithine to produce proline. Our investigations have shown that PYCRL differs from PYCR1 and 2 in the impact on cellular redox, which is a critical factor in hypoxic survival. Our data suggest that PYCRL activity is required for normal regulation of glioblastoma cell growth and the ability to deal with cellular stress, and that this enzyme may therefore represent a novel target in the treatment of these devastating tumours. Importantly, our study also begins to provide much-needed clarity over the network surrounding proline metabolism and redox maintenance.

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