LC3-Associated Phagocytosis (LAP): A Potentially Influential Mediator of Efferocytosis-Related Tumor Progression and Aggressiveness

Abstract

One aim of cancer therapies is to induce apoptosis of tumor cells. Efficient removal of the apoptotic cells requires coordinated efforts between the processes of efferocytosis and LC3-associated phagocytosis (LAP). However, this activity has also been shown to produce anti-inflammatory and immunosuppressive signals that can be utilized by live tumor cells to evade immune defense mechanisms, resulting in tumor progression and aggressiveness. In the absence of LAP, mice exhibit suppressed tumor growth during efferocytosis, while LAP-sufficient mice show enhanced tumor progression. Little is known about how LAP or its regulators directly affect efferocytosis, tumor growth and treatment responses, and identifying the mechanisms involved has the potential to lead to the discovery of novel approaches to target cancer cells. Also incompletely understood is the direct effect of apoptotic cancer cells on LAP. This is particularly important as induction of apoptosis by current cytotoxic cancer therapies can potentially stimulate LAP following efferocytosis. Herein, we highlight the current understanding of the role of LAP and its relationship with efferocytosis in the tumor microenvironment with a view to presenting novel therapeutic strategies.

Keywords: LAP; M2 macrophage activation; efferocytosis; tumor cell apoptosis; tumor immune response.

Figure 1

Clearance processes for apoptotic cell by phagocytes rely on “find me” and “eat me” signals that signal their internalization (A) “Find me” signal: apoptotic cells release signals that attract phagocytes to the site of programmed cell death. These signals include nucleotides (UTP and ATP), S1P, CX3CL1, and LPC. Phagocytes recognize the “find me” signals using cognate receptors such as P2Y2, S1PRs, CXCR3, and G2A. (B) “Eat me” signal: the apoptotic cells express “eat me” signals that allow phagocytes to recruit surface receptors and bridging molecules to identify and engulf apoptotic cells. PtdSer is a primary “eat me” signal expressed by phagocytes and is directly recognized by phagocytic receptors including BAI, TIM4, and stabilin 2. Phagocytes can employ avB3 and tyrosine kinase receptor such as Mertk bind to PtdSer indirectly through bridging molecules such as MFG-E8 and Gas-6, respectively. (C) The engulfment process: after recruitment of engulfment receptors via the CRKII-ELMO-DOCK180 complex within phagocytes, rac-1 signaling pathway is activated for phagosome formation. Once the phagosome is formed, LC3 is recruited to phagosomes (now the LAPosome) leading to lysosome-mediated digestion of the internalized apoptotic body. Degraded products release fatty acids that stimulate LXR and PPARγ for cholesterol efflux leading to the production of anti-inflammatory cytokines such as TGF-β, IL-10 and IL-13. UTP, uridine 5′ triphosphate; ATP, adenosine 5′ triphosphate; S1P, sphingosine-1-phosphate; CX3CL1, C-X3-C Motif Chemokine Ligand 1; LPC, lysophosphatidylcholine; P2Y2, purinergic receptors; SIPRs, sphingosine-1-phosphate receptors; CXCR3, C-X-C motif chemokine receptor 3; G2A, G-protein-coupled receptor; PtdSer, phosphatidylserine; BAI, brain-specific angiogenesis inhibitor 1; TIM-4, T cell immunoglobulin mucin receptor-4; avB3, alpha-v beta-3; mertk, mer proto-oncogene, tyrosine kinase; MFG-E8, milk fat globule-EGF factor 8 protein; Gas-6, growth arrest-specific 6; ELMO, engulfment and cell motility protein; DOCK, dedicator of cytokinesis; CRKII, chicken tumor virus no. 10 (CT10) regulator of kinase II; G3P, glycerol-3-phosphate guanosine; GMP, 5′-monophosphate; AMP, adenosine monophosphate; LC3, microtubule-associated protein 1A/1B-light chain 3; LXR, liver X receptor; PPARγ, peroxisome proliferator-activated receptor gamma; TGF-β, transforming growth beta; IL-10, interleukin 10; IL-13, interleukin-13. *Created with BioRender.

Figure 2

LC3-Associated phagocytosis (LAP). LAP recruitment process is triggered for degradation of the phagocytosed cargo following phagosome formation. The cargo is recognized by cell recognition receptors such as TIM-4 which causes the cargo to be engulfed in a single membrane phagosome called LAPosome. This process is initiated by recruitment of PI3KC3 complex consisting of Rubicon, vps34, beclin-1, and vps15 which enable PI3P to be localized to the LAPosomes. This stabilizes the NOX2 complex to produce ROS which is necessary to recruit LC3-II to the phagosome (LAPosome). The LAPosome then fuses with lysosomes to mature and to effective e digest the cargo. TIM-4, T-cell immunoglobulin and mucin domain family of receptors; PI3KC3 complex, Phosphatidylinositol 3-kinase catalytic subunit type 3; Rubicon, Run domain beclin-1-interacting and cysteine-rich Domain-containing protein; VPS34, Vacuolar protein sorting 34; VPS15, vacuolar protein sorting 15; PI3P, phosphatidylinositol 3-phosphate, NOX2, NADPH oxidase 2; ROS, reactive oxygen species; LC3-II, microtubule-associated protein 1A/1B-light chain 3-II. *Created with BioRender.

Figure 3

Engulfment of apoptotic tumor cells induces LAP to inhibit type I IFN response and polarize tumor associated macrophages toward the immunosuppressive M2 phenotype. This process leads to suppressed TIL function, reduced anti-tumor response and ultimately, sustained tumor survival. LAP deficiency leads to polarization of TAMs toward the pro-inflammatory M1 phenotype and stimulation of a STING-dependent type I IFN response, which enhances TIL function and expression of IFNγ to inhibit tumor growth. Taken together, these mechanisms suggest that targeting the LAP pathway may have therapeutic potential. IFN, interferon; TIL, tumor-infiltrating lymphocytes; STING, stimulator of interferon genes; IFNγ, interferon gamma. *Created with BioRender.

Figure 4

Schema of mechanism by which LAP could facilitate metastatic potential of tumors via M2 polarization. M2 promotes tumor progression through IL-10, IL-4, and TGF-β-mediated immunosuppression. They also trigger pro-survival signals such as MMPs and EGF to amplify metastatic potential of tumors by facilitating the invasion and spread of cancer cells to distant sites within the body. IL-10, interleukin-10; IL-4, interleukin-4; TGF-β, transforming growth factor-beta; MMP, matrix metallopeptidases; EGF, epidermal growth factor (74). *Created with BioRender.

Figure 5

Rubicon expression as a potential prognostic marker that has the power to predict survival outcome of some cancer patients. Representative images of Rubicon expression and survival rate of patients with breast cancer, colorectal cancer, liver cancer, prostate cancer, stomach cancer, and testicular cancer. Cancer patients overexpressing Rubicon have lower survival rate compared with patients with lower Rubicon expression. This demonstrates the need for further studies to establish prognostic values of Rubicon in different types of cancers. Credit: Human Protein Atlas, www.proteinatlas.org/humancell (95). Image available at the following URL: v19.proteinatlas.org/humancell.

Figure 6

Consequences of inhibiting factors involved in efferocytosis and LAP. (A) Macrophages interact with PtdSer externalized on apoptotic cells through efferocytic receptors on the surface of phagocytes. Many PtdSer receptors stimulate interactions to activate Rac1 and cytoskeletal arrangement for phagosome formation and internalization. Once apoptotic cells are internalized in the phagosome, LC3-recruitment process through LAP is triggered to fuse LC3 to phagosomes membranes, which facilitates phagolysosomal fusion and subsequent digestion apoptotic cell. This results in production of M2 macrophages and suppression of tumor immune activation. (B) Inhibition of PtdSer leads to defects in the phagosome formation by preventing the cytoskeletal rearrangement through Rac-1 and ELMO–DOCK180 interactions. This leads to inefficient clearance of apoptotic cells and polarization of M1 macrophages to release mediators that potentiate tumor immune responses. PtdSer, phosphatidylserine; LC3, microtubule-associated protein 1A/1B-light chain 3-II; ELMO, engulfment and cell motility protein; DOCK, dedicator of cytokinesis. *Created with BioRender.