No abstract

Metastases are the leading cause of mortality in patients with cancer. Metastasis formation requires cancer cells to adapt their cellular phenotype. However, how metabolism supports this adaptation of cancer cells is poorly defined. We use 2D versus 3D cultivation to induce a shift in the cellular phenotype of breast cancer cells. We discover that proline catabolism via proline dehydrogenase (Prodh) supports growth of breast cancer cells in 3D culture. Subsequently, we link proline catabolism to in vivo metastasis formation. In particular, we find that PRODH expression and proline catabolism is increased in metastases compared to primary breast cancers of patients and mice. Moreover, inhibiting Prodh is sufficient to impair formation of lung metastases in the orthotopic 4T1 and EMT6.5 mouse models, without adverse effects on healthy tissue and organ function. In conclusion, we discover that Prodh is a potential drug target for inhibiting metastasis formation.

SUMMARY Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2-deletion affects proliferation and metabolism in non-transformed, non-immortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Cellular proliferation depends on refilling the tricarboxylic acid (TCA) cycle to support biomass production (anaplerosis). The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC) and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis in breast-cancer-derived lung metastases compared to their primary cancers using in vivo C tracer analysis. We discovered that lung metastases have higher PC-dependent anaplerosis compared to primary breast cancers. Based on in vitro analysis and a mathematical model for the determination of compartment-specific metabolite concentrations, we found that mitochondrial pyruvate concentrations can promote PC-dependent anaplerosis via enzyme kinetics. In conclusion, we show that breast cancer cells proliferating as lung metastases activate PC-dependent anaplerosis in response to the lung microenvironment.

Growth-promoting signaling molecules including the mammalian Target of Rapamycin Complex 1 (mTORC1) drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation [1]. Glutamine is catabolyzed to α-ketoglutarate (αKG), a TCA cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate, and the second reaction occurring via glutamate dehydrogenase (GDH) or transaminases [2]. Activation of the mTORC1 pathway was previously shown to promote the anaplerotic entry of glutamine to the tricarboxylic acid (TCA) cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown [3]. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that in addition to GDH, mTORC1 could regulate GLS. Here, we demonstrate that mTORC1 positively regulates GLS and flux through this enzyme. We show that mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is critical to unwind its structured 5’ untranslated region (5’UTR). Finally, our data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN.

Metabolic transformation is a hallmark of cancer and a critical target for cancer therapy. Cancer metabolism and behavior are regulated by cell-intrinsic factors, as well as metabolite availability in the tumor microenvironment (TME). This metabolic niche within the TME is shaped by four tiers of regulation, including: 1) intrinsic tumor cell metabolism, 2) interactions between cancer and non-cancerous cells, 3) tumor location and heterogeneity, and 4) whole-body metabolic homeostasis. Here, we will define these modes of metabolic regulation and review how distinct cell types contribute to the metabolite composition of the TME. Finally, we will connect these insights to understand how each of these tiers offers a unique therapeutic potential to modulate the metabolic profile and function of all cells inhabiting the TME.

Extracellular matrix (ECM) is a major component of the local environment, i.e. the niche, that can determine cell behavior 1 . During metastatic growth, cancer cells shape the ECM of the metastatic niche by hydroxylating collagen to promote their own metastatic growth 2 , 3 . However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen because nutrients dictate which enzymatic reactions are active in cancer cells 4 , 5 . Here, we discovered that breast cancer cells rely on the nutrient pyruvate to drive collagen-based ECM remodeling in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activated collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Strikingly, inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodeling and the nutrient environment in the metastatic niche.

Little is known about the metabolism of quiescent endothelial cells (QECs). Nonetheless, when dysfunctional, QECs contribute to multiple diseases. Previously, we demonstrated that proliferating endothelial cells (PECs) use fatty acid β-oxidation (FAO) for de novo dNTP synthesis. We report now that QECs are not hypometabolic, but upregulate FAO >3-fold higher than PECs, not to support biomass or energy production but to sustain the tricarboxylic acid cycle for redox homeostasis through NADPH regeneration. Hence, endothelial loss of FAO-controlling CPT1A in CPT1A mice promotes EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress, rendering CPT1A mice more susceptible to LPS and inflammatory bowel disease. Mechanistically, Notch1 orchestrates the use of FAO for redox balance in QECs. Supplementation of acetate (metabolized to acetyl-coenzyme A) restores endothelial quiescence and counters oxidative stress-mediated EC dysfunction in CPT1A mice, offering therapeutic opportunities. Thus, QECs use FAO for vasculoprotection against oxidative stress-prone exposure.