Alessandro Rufini

Valine to glutamate substitution at residue 600 of the BRAF oncogene ( ^V600E BRAF mutation) is prevalent in human colorectal cancers with a serrated histopathology and is thought to be a founder mutation. Using a conditional knock-in mouse model we have previously demonstrated that ^V600E Braf drives crypt hyperplasia in the short term as well as shortened survival linked to increased tumour burden in the long-term. These phenotypes are associated with induction of gene signatures for E2F targets, MYC signalling, G2/M transition, canonical Wnt signalling, and cholesterol biosynthesis. Although these gene signatures are reverted by MEK inhibition, there remains a lack of understanding of the signalling pathways involved, particularly the mechanism of crosstalk between the MAPK and Wnt pathways. Here, we have examined a role for phosphorylation of GSK3αβ isoforms at residues S21/S9. By introducing homozygous knock-in mutations for Gsk3αβ S9A/S21A onto the ^V600E Braf background, we unexpectedly show a marginal effect of these mutations on further increasing crypt proliferation. However, this impact is lost in the long-term as there are no significant differences in mouse survival, tumour burden or tumour grade. Consistently, the Gsk3αβ knock-in mutations do not change the transcriptional programme induced by ^V600E Braf , except for 3 genes ( Ephx4 , Eif2b3 Ppp1r13l ) whose expression is significantly altered, potentially contributing to the short-term increase in crypt hyperplasia. Overall, our data show that therapeutic strategies targeting GSK3αβ phosphorylation at serines 21/9 are not worthwhile options for ^V600E BRAF colorectal cancers.

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we have developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are highest in liver tissue, which is also a hub for lipid production. While the loss of GSH does not cause liver failure, it decreases lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we find that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver’s balance of redox buffering and triglyceride production.

The intestinal wall represents an interactive network regulated by the intestinal epithelium, extracellular matrix (ECM) and mesenchymal compartment. Under healthy physiological conditions, the epithelium undergoes constant renewal and forms an integral and selective barrier. Following damage, the healthy epithelium is restored via a series of signalling pathways that result in remodelling of the scaffolding tissue through finely-regulated proteolysis of the ECM by proteases such as matrix metalloproteinases (MMPs). However, chronic inflammation of the gastrointestinal tract, as occurs in Inflammatory Bowel Disease (IBD), is associated with prolonged disruption of the epithelial barrier and persistent damage to the intestinal mucosa. Increased barrier permeability exhibits distinctive signatures of inflammatory, immunological and ECM components, accompanied by increased ECM proteolytic activity. This narrative review aims to bring together the current knowledge of the interplay between gut barrier, immune and ECM features in health and disease, discussing the role of barrier permeability as a discriminant between homoeostasis and IBD.

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of Braf^V600E in the intestinal tissue of an inducible mouse model. We show that Braf^V600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, Braf^V600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in Braf^V600E-mutant mice. Overall, our work unveils the long-term impact of Braf^V600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells’ proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells’ survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.

The metabolism of the non-essential amino acid L-proline is emerging as a key pathway in the metabolic rewiring that sustains cancer cells proliferation, survival and metastatic spread. Pyrroline-5-carboxylate reductase (PYCR) and proline dehydrogenase (PRODH) enzymes, which catalyze the last step in proline biosynthesis and the first step of its catabolism, respectively, have been extensively associated with the progression of several malignancies, and have been exposed as potential targets for anticancer drug development. As investigations into the links between proline metabolism and cancer accumulate, the complexity, and sometimes contradictory nature of this interaction emerge. It is clear that the role of proline metabolism enzymes in cancer depends on tumor type, with different cancers and cancer-related phenotypes displaying different dependencies on these enzymes. Unexpectedly, the outcome of rewiring proline metabolism also differs between conditions of nutrient and oxygen limitation. Here, we provide a comprehensive review of proline metabolism in cancer; we collate the experimental evidence that links proline metabolism with the different aspects of cancer progression and critically discuss the potential mechanisms involved.