Case Study 5: Understanding how metabolism influences tumour invasiveness and the microenvironment

Summary

• Elucidation of mechanisms linking metabolic re-wiring to tumour cell migration and invasion

• Stromal metabolism influences generation of invasive tumour microenvironments

Metabolic pathways driving tumour cell migration and invasion are depicted on the left, and those occurring in stromal fibroblasts are on the right. 1) SLC7A11 is upregulated in tumour cells to increase efflux of glutamate (Glut). Increased extracellular glutamate then activates the mGluR3 metabotropic receptor to promote PINK1-dependent packaging of mitochondrial DNA (mtDNA) into exosomes. mtDNA-containing exosomes activate invasion via toll-like receptor-9 (TLR9). 2) integrin-mediated mechanosensing increases creatine kinase B (CKB) to provide ATP for actin polymerisation. Stromal metabolism influences tumour cell invasiveness by synthesising lysophosphatidyl choline (LPC) which is cleaved by autotaxin to yield the pro-migratory factor lyso-phosphatidic acid (LPA). Additionally, stromal fibroblasts alter their metabolism to increase proline synthesis which, in turn, drives translation of mRNAs encoding collagens. The stiff, collagen-rich extracellular matrix (ECM) then activates mechanotransduction in tumour cells to influence their metabolism.

As tumours increase in size they encounter stresses, such as nutrient depletion and hypoxia, and they re-wire their metabolism in well-characterised ways to accommodate these stresses and to continue growing. However, in 2017, little was known about how tumour metabolism may contribute to tumour dissemination and metastasis. We, therefore, initiated a series of studies that have discovered pathways which mechanistically connect altered tumour metabolism to the invasive and migratory processes that drive metastasis. This has allowed us to dissect the molecular details of these pathways to highlight factors that may be targeted to oppose tumour cell dissemination and metabolic nodes that represent metabolic vulnerabilities of metastasising tumours.

The tumour microenvironment is metabolically stressful. This occurs because tumours consume nutrients and oxygen to generate the increased biomass that they need to grow. The significant selection pressure that is imposed on tumours by nutrient depletion and restricted oxygen supply forces them to re-wire their energy metabolism. Much attention has been devoted to characterising the metabolic re-wiring of tumours. This was primarily focused on determining how tumours continue to synthesise proteins and lipids for growth whilst maintaining redox balance, and how their metabolism can help them to evade host anti-tumour immune responses. However, metastasis is the main cause of death from cancer and, prior to 2019, the understanding of how altered tumour cell metabolism might influence metastasis was less clear. With its expertise in metabolomics and established track-record in vivo and ex vivo approaches to studying invasion and metastasis, the CRUK Beatson Institute is uniquely positioned to investigate the relationship between tumour metabolism and metastasis and to address how the cellular machinery that drives invasive behaviour might be mechanistically linked to the means of energy production in the cell.

A key initial step in metastasis is the ability of tumour cells to breach the basement membrane and to migrate away from the primary tumour site. This is dictated, not only by the migratory capabilities of tumour cells, but also by the nature of the surrounding extracellular matrix (ECM) that is made, primarily, by fibroblasts in the evolving tumour stroma. We have led a cluster of seminal studies that define how the migratory machinery of tumour cells, and the ability of carcinoma-associated fibroblasts (CAFs) to manufacture ECM in the tumour, are mechanistically connected to, and driven by, the metabolic rewiring that accompanies tumour progression.

Metabolic re-wiring drives tumour cell migration and invasion

Tumour cells evoke a cytoprotective programme in response to metabolic stress and central to this is upregulation of the SLC7A11 cystine-glutamate antiporter. SLC7A11 imports cystine for glutathione synthesis to help maintain redox balance and this occurs at the expense of glutamate export. The Norman, Blyth, Tait and Gammage laboratories, in collaboration with David Sumpton in the Metabolomics facility, have shown that the combination of upregulated glutaminolysis and expression of SLC7A11 leads to accumulation of glutamate in the extracellular space. This, in turn, activates a metabotropic glutamate receptor (mGluR3) to increase Rab27-dependent trafficking of a transmembrane matrix metalloprotease (MT1-MMP) to the plasma membrane and PINK1-dependent release of exosomes containing mitochondrial DNA from tumour cells. Increased MT1-MMP trafficking, and release of mitochondrial DNA conspire to drive tumour invasiveness allowing breast cancers to breach their basement membrane and invade the surrounding tissue (1,2). Importantly, a drug that blocks the mGluR3 glutamate receptor opposes metastasis of breast cancer to the lung.

In addition to being metabolically stressed, solid tumours – particularly pancreatic adenocarcinoma (PDAC) – are often mechanically very rigid, and tumour stiffness is known to be a driver of invasion and metastasis. Indeed, recent ground-breaking work from the Morton and Jørgensen labs has indicated that it is necessary to recapitulate the rigid mechanics of the ECM to faithfully represent PDAC phenotypes in ex vivo organoid culture (3). To exploit this phenomenon, the Machesky and Maddocks groups combined bioengineering with metabolomics, which enabled them to identify a new pathway linking tumour stiffness to metabolism via the creatine-phosphagen ATP recycling system. Indeed, mechanotransduction evoked by stiff ECM upregulates creatine kinase B, which plays a key role in providing energy for the cell’s migratory machinery to promote invasive migration and metastasis of PDAC to the liver (4).    

Stromal metabolism influences ECM synthesis and deposition

The make-up of the tumour stroma is key to disease progression and metastasis, and the Morton and Sansom groups with Claus Jørgensen’s group in Manchester have recently shown how the complexity of the stroma dictates outcomes in PDAC (5). Although the possibility of metabolic crosstalk between stromal fibroblasts and PDAC cells had previously been considered, it was not until 2019 – when the Kamphorst and Norman groups profiled the metabolism of pancreatic stellate and tumour cells – that this was established as important to disease progression. Indeed, these groups demonstrated that activated pancreatic stellate cells are major synthesisers of lysophosphatidyl cholines (LPCs), which are then released into the tumour microenvironment. These LPCs are then hydrolysed extracellularly by the lysophospholipase, autotaxin to produce lysophosphatidic acid (LPA), which in turn signals to the cancer cells to drive invasive migration (6). This work is the first to highlight the metabolic crosstalk between distinct fibroblast subtypes and cancer cells and is key to understanding the way in which lipid metabolism fuels invasiveness.

Tumour stiffness is a particularly important driver of metastasis and, as we have demonstrated (see above), this can be due to the influence of rigidity on tumour cell metabolism. But, to what extent does the metabolism of the stroma influence tumour stiffness?  The Zanivan, Blyth, Miller, Kamphorst and Tardito groups recently addressed this important question by profiling the metabolism of CAFs. They found that these cells have elevated synthesis of acetyl-CoA (via pyruvate dehydrogenase), which leads to markedly increased production of proline. Proline is an important building block for collagen, and these groups found that proline produced via this route was incorporated into the ECM to generate a stiff, pro-invasive tumour microenvironment conducive to tumour invasion and progression (7). Moreover, this important study provides evidence that pharmacological targeting of the proline synthesis pathway halts the production of a pro-invasive tumour ECM.

References

1. Dornier E et al. Glutaminolysis drives membrane trafficking to promote invasiveness of breast cancer cells. Nat Commun. 2017; 8: 2255
2. Rabas N et al. PINK1 drives production of mtDNA-containing extracellular vesicles to promote invasiveness. J Cell Biol. 2021; 220: e202006049
3. Below CR et al. A microenvironment-inspired synthetic three-dimensional model for pancreatic ductal adenocarcinoma organoids. Nat Mater. 2022; 21: 110-9
4. Papalazarou V et al. The creatine-phosphagen system is mechanoresponsive in pancreatic adenocarcinoma and fuels invasion and metastasis. Nat Metab. 2020; 2: 62-80
5. Hutton C et al. Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity. Cancer Cell. 2021; 39: 1227-44 e1220
6. Auciello FR et al. A stromal lysolipid-autotaxin signaling axis promotes pancreatic tumor progression. Cancer Discov. 2019; 9: 617-27
7. Kay EJ et al. Cancer-associated fibroblasts require proline synthesis by PYCR1 for the deposition of pro-tumorigenic extracellular matrix. Nat Metab. 2022; 4: 693-710