Importance of mTOR complex 2 (mTORC2) and AMPK in neutrophils to Melanoma progression

Abstract

Neutrophils are considered potent inflammatory cells. Besides, they can recruit other cells to the site of inflammation and are able to orchestrate the immune response through the communication with macrophages, dendritic cells or adaptive immune cells by cell-cell interaction or soluble mediators. Thus, neutrophils are plastic and phenotypically diverse cells acting not in acute but also in chronic diseases, as Cancer. Different tumor types have an abundant neutrophilic infiltrate. These neutrophils can act promoting or inhibiting the tumor growth. It is depending on the signals from the tumor microenvironment. Several subpopulations of neutrophils can be found in the tumor microenvironment, such as N1, N2 and G-MDSCs (granulocytic myeloid-derived suppressor cells). N2 and G-MDSC are associated to tumor progression for inhibiting T lymphocyte proliferation. One of the mechanisms used to neutrophils to inhibit the proliferation of T lymphocytes is the exacerbated ROS, NO and arginase-1 production. Thus, pro-tumoral neutrophils have an oxidative metabolism allowing them oxidizing fatty acids to generate NADPH, increasing the production of oxidants. It is well known that many metabolic signaling pathways are crucial in the tumor context. In this sense, mTOR signaling pathway has been spotlighted during the past years. This pathway has two main complexes, mTORC1 and mTORC2. mTORC1 inhibition in tumor cells has been associated to preventing tumor growth by inhibiting cells proliferation. In this sense, the negative regulators of mTORC1 have been studied for having anti-tumoral function. One of most important mTORC1 negative regulators is the protein kinase. Drugs like metformin that activates AMPK, has been targeted as anti-tumoral drugs. Due to the lack of a selective inhibitor, mTORC2 is not well studied in the tumoral context. So, we hypothesized that AMPK and mTORC2 acting regulation the metabolism and the effector functions of neutrophils, impacting on Melanoma progression. Our preliminary results show that AMPK deletion in neutrophils increase ROS and NETs production, leading to a pro-tumoral (N2) profile of neutrophils (improving the Melanoma progression). On the other hand, Rictor-deleted neutrophils generate less ROS and NETs, possibly, leading to an anti-tumoral neutrophils profile (N1). Using cre-lox mice where AMPK or Rictor (mTORC2) are deleted specifically in myeloid cells, we will evaluate the importance of theses molecules to the metabolism and effector functions of neutrophils and how it affects the Melanoma cells (B16-F10) metabolism and progression. To evaluate it, we will perform co-culture assays and we will check if the presence of neutrophils alters invasion, proliferative and metabolic characteristics in tumor cells. We will also evaluate the metabolism and effector functions of neutrophils after contact with tumor cells and the impact of AMPK or Rictor during this process. Using in vivo model (subcutaneous or metastatic models), we will evaluate the neutrophilic infiltrate in the tumor and check the disease progression in the animals deleted to AMPK or Rictor. At last, using blood samples from Melanoma patients, we will evaluate the metabolic profile and effector functions of the neutrophils and mTOR pathway activation. We will correlate it to the tumor phase and the in vitro results. In this sense, the mTOR pathway study and its regulators in immune cells, mainly the neutrophils, in a tumor context is crucial to reach new therapeutic strategies. (AU)