Upcycling the TCA cycle—rewiring tumour-associated fibroblasts

Alterations to the extracellular matrix have long been associated with cancer progression and therapeutic resistance. Schwörer et al. describe a mechanism whereby fibroblasts reroute metabolites to fuel the demands of collagen synthesis, leading to cancer progression.

The tumour microenvironment (TME) is a complex and unique meshwork of cell types that coexist within the extracellular matrix (ECM), a scaffold comprising proteins and glycoproteins. Collagen is a major component of the ECM, and aberrant deposition of collagen is a known factor for tumour progression^1,2. Previous studies performed in vitro have shown that transforming growth factor-β (TGFβ) promotes ECM synthesis, and studies by Schwörer et al. and others have mechanistically attributed this to an increase in glucose and glutamine uptake^3,4,5,6. However, the physiological abundances of these nutrients in human tissue and the TME are significantly less than the concentrations found in typical cell culture conditions⁷. In the latest issue of Nature Metabolism, Schwörer et al. utilize in vivo tumour models and demonstrate that fibroblasts rely on the activity of pyruvate carboxylase (PC) to drive the TCA cycle to maintain protein translation and collagen production, a process dependent on de novo glutamine synthesis⁸. Together, these findings describe a mechanism by which tumour-associated fibroblasts sustain aberrant ECM production in an environment with limited metabolic resources, highlighting a potential area for the development of novel therapies.

It has long been appreciated that fibroblasts play an integral role in the composition of the tumour microenvironment ECM via the synthesis of proteins such as collagen, fibronectin and matrix metalloproteases influencing the deposition and degradation of the scaffold⁹. Observations made nearly 40 years ago began linking the recruitment and activation of fibroblasts in human cancers to ‘migration factors’, which were later identified to be growth factors such as TGFβ^3,10,11. More recent efforts have focused on delineating the mechanism by which TGFβ supports the bioenergetic demand that tumour-associated fibroblasts require for the excessive production of ECM factors^4,5,6,12. A previous study⁶ led by the Thompson lab showed that TGFβ supports protein translation by regulating the expression of genes responsible for glucose and glutamine uptake through the activity of the transcription factor SMAD4. The observed increase in glucose and glutamine oxidation proceed to fuel mitochondrial respiration, providing an abundance of ATP and metabolic precursors⁶. Employing metabolic tracing, Schwörer et al. treated fibroblasts with TGFβ and confirmed that TGFβ increased glutamine incorporation into proline, enhanced the expression of genes involved in proline synthesis, and increased glucose incorporation into serine and glycine⁶. In this study, they also observed that TCA cycle activity and mitochondrial respiration were required for TGFβ-stimulated proline synthesis, which they followed up on in a subsequent study where they discovered that generation of mitochondrial nicotinamide adenine dinucleotide phosphate (NADPH) is required for the synthesis of proline from glutamate, aiding collagen production^6,12. These studies comprehensively characterized how fibroblasts respond to TGFβ and the cascade of metabolic rewiring that occurs to drive collagen synthesis.

In the current study, Schwörer et al. begin by investigating how fibroblasts respond to various concentrations of glutamine available in the culture medium in the presence or absence of TGFβ. These experiments found that, under poor glutamine availability, the proliferation of TGFβ-treated fibroblasts halted and cells were unable to produce collagen I. Next, they sought to understand why TGFβ-stimulated fibroblasts experienced inhibited collagen production in response to limited glutamine availability. The authors elegantly implemented a tRNA charging assay previously developed by the Thompson lab¹³ as a way to measure active protein synthesis and observed a remarkable decrease in charged glutamine-tRNA in TGFβ-stimulated fibroblasts cultured in low glutamine. Metabolite analysis revealed an associated reduction in TCA cycle intermediates, as well as glutamate, aspartate and asparagine, indicating that mitochondrial metabolism was impaired. Notably, restoration of these features was achieved by supplementing with cell-permeable glutamate and α-ketoglutarate (αKG). Using pharmacological and genetic approaches to suppress glutamine synthetase (GLUL), the key enzyme converting glutamate to glutamine, they demonstrated that de novo glutamine synthesis is a critical lifeline to replenish the TCA cycle in a nutrient-poor environment for TGFβ-stimulated fibroblasts.

Pyruvate is the other major carbon source for TCA anaplerosis. Stimulation with TGFβ resulted in transcriptional repression and impeded function of pyruvate carboxylase, the enzyme responsible for converting pyruvate to oxaloacetate for entry into the TCA cycle. Overexpression of PC was sufficient to restore glucose-derived carbons in the TCA cycle, increase protein translation and collagen production and increase fibroblast proliferation. Strikingly, an enrichment in SMAD4 binding motifs, as well as an increased presence of SMAD4 at the PC promoter region and transcriptional start site (TSS), revealed TGFβ-dependent downregulation of PC to be directed by SMAD4. Deletion of PC from fibroblasts reduced collagen I expression, which could be rescued with the addition of glutamate or αKG. Though PC activity is tightly linked to glucose oxidation, glucose tracing in PC-deleted cells showed a significant reduction in glucose-labelled glutamine, which suggests that PC anaplerotic activity does support de novo glutamine synthesis. Further, PC deletion resulted in reduced histone acetylation (H3K27ac), a mark associated with active transcription, and increased methylation (H3K27me3), a repressive mark, both at the Col1a1 locus, highlighting the functional connections between metabolism and epigenetic programming.

A key finding of the paper was that lactate was being used for anaplerosis via PC activity. Functionally, lactate was being incorporated into collagen present in the ECM deposited by the fibroblasts on their cell culture plates. In a co-culture spheroid growth model, the authors deleted either PC or GLUL from primary pluripotent stem cells (PSC), which differentiate into cells of the mesenchymal lineage such as fibroblasts. Finally, they used in vivo breast and pancreatic cancer models in which they co-injected tumour cells together with PSCs or myofibroblasts (MFB) that harboured PC or GLUL deletion into the flanks of both immunocompromised and immunocompetent recipients. The deletion of PC or GLUL was sufficient to reduce tumour growth and impair the ability to synthesize proline and collagen in the TME.

Schwörer et al. establish a novel mechanism by which fibroblasts can navigate metabolic stress, utilizing various fuel sources to feed the TCA cycle and modifying epigenetic marks to regulate collagen production (Fig. 1). Having identified a metabolic dependency in fibroblasts that translates to their ability to support tumour growth, future studies aimed at testing the clinical relevance will be essential to the development of this area. The extent to which these fibroblasts contribute to tumour maintenance as metabolic reservoirs is also an area for future consideration. Given the focus on the tumour microenvironment, future experiments will need to examine the extended cellular components of the TME. The findings described by Schwörer et al. have paved the way for further interrogation of fibroblast metabolism and discovery of other metabolic dependencies that could be exploited to treat fibrotic disease.

Fig. 1: Fibroblasts upregulate pyruvate carboxylase to obtain metabolic flexibility and sustain collagen production in a nutrient-poor tumour microenvironment.

In healthy tissue, fibroblasts generally exist in a much less dense environment where the demands for collagen and ECM synthesis are met with an abundance of nutrient resources such as glutamine. By contrast, the tumour microenvironment forces fibroblasts to compete for metabolic resources with cancer and immune cells in a space where nutrients are often scarce due to poor vascularization. To overcome these pressures, TGFβ-stimulated cancer-associated fibroblasts transcriptionally upregulate pyruvate carboxylase via the direct activity of SMAD4. By increasing expression of pyruvate carboxylase, fibroblasts in the TME can utilize glucose- or lactate-derived pyruvate to fuel the TCA cycle and support de novo glutamine synthesis, sustainably providing precursors for amino acid synthesis and collagen production.