Stromal-derived lactate activates amoeboid motility by engaging the endocannabinoid receptor GPR55 in a prostate cancer cell model

The tumor microenvironment (TME) is a dynamic environment that surrounds tumors, composed of infiltrated/ resident host cells, the extracellular matrix (ECM), and soluble factors. Among the latter, lactate is one of the most abundant metabolites produced by the high glycolytic activity of tumor and stromal cells, which fosters cell anabolic processes conducive to tumor development. Lactate plays a key role in promoting characteristics associated with tumor aggressiveness, including cell invasiveness, as demonstrated in prostate cancer (PCa). Furthermore, its newly identified function as a ligand targeting GPCRs, particularly GPR81, is noteworthy. The study of the TME has expanded today to include the Endocannabinoid system (ECS), a cell network constituted of endocannabinoids (e.g., anandamide, 2-arachidonoylglycerol), cannabinoid receptors (CBRs), and enzymes. Across various types of tumors, the ECS spans both cancer cells and accessory cells, significantly influencing tumor cells' proliferation, survival, and metastasis, and it serves as a ‘cornerstone’ in the processes of angiogenesis, immune response, and stromal cells’ functionality. However, the system's involvement in the intricate tumor-stroma interplay remains unclear. Given the aforementioned considerations, our study aimed to establish a connection between the pro-tumor role of lactate and the functionality of the ECS, focusing on an experimental model of PCa. From the results, we observed that lactate, at a concentration equivalent to those measured in prostate tumor biopsies (20 mM), up-regulated GPR55, without inducing positive alterations in the expression of CB1-2R and TRPV1. GPR55 is a non-canonical CBR, with a weak affinity for endocannabinoids, and primarily binding lysophosphatidylinositol (LPI). Lactate stimulation at a low dose (2.5 mM) and rapid timeframes (15 minutes) induced phosphorylation of MCL2 in PCa cells, an acknowledged pathway downstream of GPR55. The blockade of the receptor with a selective inhibitor (ML193) prevented lactate-induced MLC2 activation. The low dose and the rapidity with which lactate activated a receptor led us to suppose that lactate may exploit a “hormonal-like” role towards GPR55, as found for GPR81. Across different tumor types, GPR55 mainly holds a pro-tumor role. In our experimental model, the exposure of PCa cells to lactate promoted migratory skills by engaging GPR55, as demonstrated by the treatment with two GPR55 selective inhibitors, ML193 and CID 16020046. The usage of the preferential agonist for GPR55, LPI, confirmed the involvement of GPR55 in the phenotype elicited by lactate in PCa cells. Activation of Rho A is a GPR55-related signaling that is likely associated with cell motility. In PCa cells, lactate activation of GPR55 up-regulated Rho A functionality, strengthening the engagement of GPR55 in lactate-sustained cell invasiveness. MLC2 phosphorylation and Rho A activation are typical events that characterize amoeboid motility, a cell movement that doesn’t necessarily rely on metalloproteinase (MMP) activity, such as mesenchymal motility. Delving into a potential involvement of the lactate/ GPR55 axis in cell invasiveness phenomena connected to the amoeboid movement, we used: the Rho kinase inhibitor (CT04) and broad-spectrum MMPs inhibitor (Marimastat). From the results we obtained: I) CT04 decreased lactate-induced PCa cell invasion similarly to what was achieved with ML193; and II) Marimastat didn’t significantly decrease PCa cell motility. Therefore, given the strong efficacy of CT04 and the low capacity of Marimastat in preventing cell invasion, we assumed lactate was involved in amoeboid-like invasive skills of PCa cells, through the GPR55/ Rho A pathway. Overall our findings suggested a pivotal role of lactate in modulating GPR55 expression and activity in PCa cells, thus promoting invasive skills through the acquisition of amoeboid-like traits.