Neuronal glutamate-mediated pancreatic cancer progression through neuro-cancer synapses

神经侵袭(NI)是影响胰腺癌(PCa)预后的重要因素之一。在NI期间，PCA细胞附着在胰腺内神经上，并利用它们作为传播途径。然而，NI的分子机制尚未完全阐明。最近的研究表明，在中枢神经系统中，胶质瘤细胞或转移性乳腺癌细胞可以与神经元突触形成假三方突触，以类似于兴奋性突触结构中的方式用L谷氨酸为自己提供燃料。这种谷氨酸-NMDA轴和神经系统外的伪三方突触的存在，即在像PCa这样的周围性癌症中，还没有被证明。在这里，我们打算探索类似的谷氨酸能神经元对癌细胞的谷氨酸-NMDAR信号的输入是否促进了癌细胞的生长并促进了前列腺癌的NI。在我们的初步研究中，我们在CCLE数据库中描绘了人和小鼠前列腺癌细胞系的谷氨酸受体图谱，并将这种模式与神经或非神经侵袭性前列腺癌细胞系相关联。我们发现谷氨酸受体亚型GluN2D(由GRIN2D编码)明显参与了L-谷氨酸或背根神经节条件培养液(CM)介导的神经侵袭性胰腺癌细胞的迁移和侵袭。然而，在非神经侵袭性胰腺癌细胞中，GluN2D拮抗剂和GRIN2D siRNA处理后，这些表型特征可以完全逆转。与DRG或DRG CM共同培养后，谷氨酸-GluN2D-NMDAR信号转导分子被激活和上调。在神经元和PCa癌细胞的共同培养中，GRIN2D、GRIN1和NMDAR相互作用伙伴PSD95在暴露于神经元的神经侵袭性PCa细胞中上调，而Synaptobrevin-1和vGlut-2显著存在于从神经元延伸并与PCa细胞突触的轴突上。这一发现在非神经侵袭性前列腺癌细胞中没有观察到。在机制上，谷氨酸或DRG CM处理导致转录因子EZH2通过EZH2-E2F1-Rb途径在Grin2d启动子上浓缩。我们得出结论，神经元通过GluN2D介导的谷氨酸-NMDAR信号促进PCA向神经元的迁移，该信号受上游EZH2-E2F1-Rb通路的调节。突触触发的PCA迁移之间的这种分子机制代表了一种新的靶向途径，我们希望在本文提出的体内靶向方法中进一步探索这一途径。此外，我们将评估通过谷氨酸-GluN2D信号与PCA细胞相互作用的DRG神经元的电活动，并将通过电子显微镜原位检测人类PCA中可能的神经癌突触。总之，这项拟议的研究将促进我们对神经元-癌症相互作用的理解，并提供潜在的可操作靶点，以对抗PCa中的NI。

Neural invasion (NI) is one of the strongest prognostic factors in human pancreatic cancer (PCa). During NI, PCa cells adhere to intrapancreatic nerves and exploit them as routes of spread. The molecular mechanisams of NI have, however, not yet been fully elucidated. Recent studies have shown that in the central nervous system, glioma cells or metastatic breast cancer cells can form pseudo-tripartite synapses with neuronal synapses to fuel themselves with L-glutamate in a manner similar to that found within an excitatory synapse structure. The presence of such a glutamate-NMDA axis and of pseudo-tripartite synapses outside the nervous system, i.e., in a peripheral cancer like PCa, has not yet been demonstrated. Here, we intend to explore whether a similar glutamatergic neuronal input over the glutamate-NMDAR signaling into the cancer cells fosters their growth and promotes NI in PCa. In our preliminary study, we profiled the glutamate receptor profile of human and murine PCA cell lines on the CCLE database and correlated this pattern to the neuro-or non-neuro-invasive PCA cell lines. We found that the glutamate receptor subtype GluN2D (encoded by GRIN2D) was explicitly involved in cell migration and invasion of the neuroinvasive pancreatic cancer cells mediated by L-Glutamate or DRG (dorsal root ganglion) conditioned medium (CM). These phenotypic features could be completely reversed after GluN2D antagonist and GRIN2D siRNA treatment, however, not in non-neuroinvasive pancreatic cancer cells. The transduction molecules of Glutamate-GluN2D-NMDAR signaling were activated and up-regulated by co-culture with DRG or DRG CM. In co-cultures of neurons and PCa cancer cells, GRIN2D, GRIN1, and the NMDAR-interaction partners PSD95 were upregulated in neuro-invasive PCa cells exposed to neurons, whereas synaptobrevin-1 and vGlut-2 were notably present on axons that extended from neurons and synapsed on PCa cells. This finding was not observed in non-neuroinvasive PCa cells. Mechanistically, glutamate or DRG CM treatment led to the enrichment of the transcription factor EZH2 on the Grin2d promotor through the EZH2-E2F1-Rb pathway. We conclude that neurons promote the migration of PCA towards neurons in a glutamate-fueled manner via GluN2D mediated Glutamate-NMDAR signaling, which is regulated by the upstream EZH2-E2F1-RB pathway. This molecular machinery between synapse-triggered PCA migration represents a novel targetable pathway, which we want to explore further within the herein proposed in vivo targeting approaches. Furthermore, we will assess the electrical activity in DRG neurons that interact with PCa cells over the glutamate-GluN2D signalling and will detect the putative neuro-cancer synapses via electron microscopy in human PCa in situ. Collectively, the proposed study will advance our understanding of neuron-cancer interactions and provide potential actionable targets against NI in PCa.