The role of glutaminolysis in providing metabolites to support tumour growth is well-established, but the involvement of glutamine metabolism in invasive processes is yet to be elucidated. Here we show that normal mammary epithelial cells consume glutamine, but do not secrete glutamate. Indeed, low levels of extracellular glutamate are necessary to maintain epithelial homoeostasis, and provision of glutamate drives disruption of epithelial morphology and promotes key characteristics of the invasive phenotype such as lumen-filling and basement membrane disruption. By contrast, primary cultures of invasive breast cancer cells convert glutamine to glutamate which is released from the cell through the system Xc- antiporter to activate a metabotropic glutamate receptor. This contributes to the intrinsic aggressiveness of these cells by upregulating Rab27-dependent recycling of the transmembrane matrix metalloprotease, MT1-MMP to promote invasive behaviour leading to basement membrane disruption. These data indicate that acquisition of the ability to release glutamate is a key watershed in disease aggressiveness.
Radiotherapy is one of the most effective approaches to achieve tumour control in cancer patients, although healthy tissue injury due to off-target radiation exposure can occur. In this study, we used a model of acute radiation injury to the lung in the context of cancer metastasis, to understand the biological link between tissue damage and cancer progression. We exposed healthy mouse lung tissue to radiation prior to the induction of metastasis and observed a strong enhancement of cancer cell growth. We found that locally activated neutrophils were key drivers of the tumour-supportive preconditioning of the lung microenvironment, governed by enhanced regenerative Notch signalling. Importantly, these tissue perturbations endowed arriving cancer cells with an augmented stemness phenotype. By preventing neutrophil-dependent Notch activation, via blocking degranulation, we were able to significantly offset the radiation-enhanced metastases. This work highlights a pro-tumorigenic activity of neutrophils, which is likely linked to their tissue regenerative functions.
The cystine-glutamate antiporter, xCT, supports a glutathione synthesis program enabling cancer cells to cope with metabolically stressful microenvironments. Up-regulated xCT, in combination with glutaminolysis, leads to increased extracellular glutamate, which promotes invasive behavior by activating metabotropic glutamate receptor 3 (mGluR3). Here we show that activation of mGluR3 in breast cancer cells activates Rab27-dependent release of extracellular vesicles (EVs), which can transfer invasive characteristics to “recipient” tumor cells. These EVs contain mitochondrial DNA (mtDNA), which is packaged via a PINK1-dependent mechanism. We highlight mtDNA as a key EV cargo necessary and sufficient for intercellular transfer of invasive behavior by activating Toll-like receptor 9 in recipient cells, and this involves increased endosomal trafficking of pro-invasive receptors. We propose that an EV-mediated mechanism, through which altered cellular metabolism in one cell influences endosomal trafficking in other cells, is key to generation and dissemination of pro-invasive microenvironments during mammary carcinoma progression.
Glutamine metabolism is well-established to contribute to cancer cell growth and proliferation by providing a source of nitrogen for nucleotide and amino acid biosynthesis as well as TCA cycle intermediates. There is also accumulating evidence that glutamine metabolism may contribute to metastasis although mechanistic links to tumour cell migration and invasion remain unclear. We have generated a number of highly invasive primary cell lines from the polyoma middle-T genetically engineered mouse model of breast cancer (MMTV-PyMT) and found that withdrawal of glutamine from these cells reduces not only their proliferation, but also their invasive migration into 'stroma-like' preparations of fibroblast-derived extracellular matrix. Our metabolomic analyses indicate that invasive MMTV-PyMT cells actively secrete glutamate, a product of glutamine metabolism, into the extracellular milieu. Moreover, addition of glutamate is sufficient to restore invasiveness (but not cell growth or proliferation) to glutamine-starved MMTV-PyMT cells. We have pursued these findings by investigating the role played by plasma membrane receptors for glutamate in cell migration and invasion in PyMT cells and in MDA-MB-231 triple negative breast cancer cells. We provide evidence that glutamate generated within the cell by deamidation of glutamine leaves the cell via the xCT antiporter to activate the GRM3 metabotropic glutamate receptor at the cell surface. This, in turn, suppresses adenylate cyclase activity to prevent protein kinase A activation and to drive an invasive programme. Indeed, knocking out GRM3 with CRISPR technology or inhibition using a selective GRM3 antagonist (LY341495) is sufficient to oppose invasiveness without compromising proliferation. Conversely, a specific GRM3 agonist (LY354740) drives invasiveness without increasing proliferation. Consistently, treatment with LY341495 was sufficient to abrogate lung colonisation following tail vein injection whilst tumour growth after orthotopic injection was unaffected. Our results provide a mechanistic link between glutamine metabolism and invasion and identify GRM3 as a potential therapeutic target in breast cancer. Citation Format: Macpherson IR, Dornier E, Rabas N, Rainero E, Norman JC. Glutamine metabolism drives breast cancer invasion by providing a source of extracellular glutamate to activate the GRM3 metabotropic glutamate receptor [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-06.
Metastatic relapse can occur months to years after the cancer is first diagnosed, and it relies on the reactivation of disseminated tumour cells (DTCs) that have lied dormant in secondary organs. The acquisition and maintenance of a dormancy phenotype are tightly regulated by the interaction of DTCs with their microenvironment. In the bone, the dormant program activated in metastatic competent cells and how their reactivation is induced are largely unknown. We here present a strategy to engineer a mouse-to-mouse extramedullary (EM) bone, which faithfully recapitulates endogenous bone and that can induce the acquisition of dormancy in highly metastatic breast cancer cells. By characterising the dormant state of metastatic cells, we found that they persist in two distinct transcriptional states: a more abundant quiescent and a less abundant proliferative state. The local environment influences the ratio between the two states. We found that an inflammatory mediator expressed by neutrophils progenitors, HMGB2, can contribute to nudging DTCs into an increased proliferation. Indeed, waves of emergency granulopoiesis expanding the neutrophils pool caused by inflammatory events such as intestinal colitis, expanded the proliferative pool of DTCs without resulting in a direct outgrowth. However, these events, progressively augmented the amount of DTCs in the bone, increasing the chance of overt metastasis. Therefore, this study proposes a piecemeal model of metastatic reactivation, where subsequent inflammatory events challenging the bone environment progressively increase the likelihood of outgrowth over time as opposed to a sudden switch from dormancy to metastasis.
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