Post by Stephanie Williams
What's the science?
Cancer cells in solid breast tumors commonly seed cells in the brain and acquire the ability to metastasize (grow in an organ distant from the original tumor). Breast-to-brain metastasis is well characterized, but it is not known why this particular form of metastasis is common. Previous research has identified a signaling pathway that may be involved - a type of glutamate receptor called N-methyl-D-aspartate (NMDA), that is activated by the excitatory neurotransmitter glutamate. Glutamatergic signaling via NMDA receptors is known to support neuroendocrine and ductal pancreatic cancer tumor growth. It has not yet been investigated whether similar glutamatergic signaling is responsible for breast-to-brain metastatic growth. This week in Nature, Zeng and colleagues identify a mechanism that explains how breast cancer cells can position themselves in existing synapses (spaces between neurons in the brain) to facilitate metastatic growth in the brain.
How did they do it?
The authors performed a series of experiments in human and mouse cell lines to assess the role of glutamate-mediated signaling in human breast to brain metastasis. 1) First, they examined the expression of different glutamate receptor subunit genes across human cancer types, and ultimately focused on breast cancer. To understand the relationship between breast-to-brain metastasis and the NMDA receptor, the authors investigated whether a particular subunit of the receptor, a protein called ‘GluN2B’, was phosphorylated at particular sites in brain metastasis tissue versus in primary breast tissue (no metastasis). Phosphorylation at particular sites can allow the NMDA receptor to reach the surface of cells, where it can participate in signaling with glutamate. The authors compared the ratio of phosphorylated to total GluN2B protein in primary breast cancer tissue with that from breast-to-brain metastatic cell tissue. The results of these analyses led the authors to focus on the GluN2B subunit of the NMDA receptor for further analyses. 2) They confirmed NMDA GluN2B-mediated signaling was functional in the breast-to-brain metastatic cells by applying L-glutamate (known to activate the receptor) and analyzing whether phosphorylation of the GluN2B subunit had occurred. They also imaged the breast-to-brain metastatic cells while applying either NMDA or glutamate exogenously and looking for responses typical of active cells (elevated intracellular calcium & single-channel currents), to confirm the signaling was mediated by NMDA. 3) To understand where the activating molecule, L-glutamate (that activated the NMDA receptor subunit), was originating, the authors performed analyses in different cell lines: they stained sections of both tissue around the lesions containing the metastatic cells and normal tissue to investigate whether the cells were absorbing glutamate from glutamatergic synapses. They used stimulated emission depleted super-resolution microscopy to image the synapses. The authors also used electron microscopy to further examine the structure of the interactions between the cells and existing synapses. 4) Finally, the authors identified the stage at which the GluN2B-mediated signaling contributed to brain metastasis. To confirm that NMDA receptor signaling was important for the proliferation of the cells, the authors disrupted signaling at different stages of tumor development in cells in which NMDA receptor signaling could be disrupted (DOX-inducible knockdown).
What did they find?
The authors found that GluN2B-mediated signaling in cancer cells in the brain is activated by interactions between metastatic cells and neurons in the brain. From their analyses of different receptor subunit genes in humans, the authors found that tumors cells in humans exhibited higher NMDA receptor expression scores versus other glutamate receptors, and that gene expression encoding GluN2B was high in a type of breast cancer with a poor prognosis. When the authors compared several human breast cancer cell lines in mice, they found that GluN2 was upregulated in the breast-to-brain metastatic line. When mice were inoculated with cells from this line at different locations in the body, brain metastases were highly stained for phosphorylated GluN2B compared to the breast and lung.
Super-resolution microscopy revealed that metastatic cell puncta (processes; stained with luciferase) were in close proximity to presynaptic neurons (stained with vGlut2) and NMDA receptors (stained with pGluN2B). The authors also observed that the metastatic tissue exhibited increased expression of a key postsynaptic signal-transducing protein as well as other markers including neuroligin, which facilitates adhesion and psuedo-synapse formation between cells. These findings suggest that metastatic human breast cancer cells access glutamate the same way that neuronal cells do (by forming synapses with neurons), and that upregulated NMDA receptors may play a role in the brain-metastatic proficiency of the breast-to-brain metastatic cells. The electron microscopy images of the metastatic tissue revealed “finger-like processes” that extended from the breast-to-brain metastatic cells toward excitatory synapses. The authors note that extended process from the breast-to-brain metastatic cells did not disrupt the pre-post neuronal synapse, but was similar to the position usually occupied by astrocytes. The results from the 3D electron microscopy suggest that breast-to-brain metastatic cells are positioned in the pseudo-tripartite synapse (i.e. cells were associated with both pre and postsynaptic membrane) and access the glutamate secreted by presynaptic neurons. When the authors disrupted NMDA receptor signaling, they found that the proliferation of the metastatic cells was disrupted, and that restoring NMDA signaling increased the proliferation of the breast-to-brain metastatic cells again. These findings suggest that GluN2B-NMDA receptor signaling was not essential for metastatic seeding, but rather promoted colonization and tumor growth in the brain.
What's the impact?
The study identified a signaling pathway that mechanistically explains metastatic tumor growth in the brain. The findings show that cancerous cells position themselves next to glutamatergic synapses in the brain, allowing them to access glutamate which ultimately promotes metastasis via NMDA receptor signaling. This finding will enable future research to identify specific vulnerabilities in the NMDA-related metastatic pathway that could be targeted to block brain metastasis without harming nearby neurons.
Zeng et al. Synaptic Proximity Enables NMDAR Signaling To Promote Brain Metastasis. Nature (2019). Access the original scientific publication here.