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Abstract: Human cancer cells operate a variety of effective molecular and signaling mechanisms which allow them to escape host immune surveillance and thus progress the disease. We have recently reported that the immune receptor Tim-3 and its natural ligand galectin-9 are involved in the immune escape of human acute myeloid leukemia (AML) cells. These cells use the neuronal receptor latrophilin 1 (LPHN1) and its ligand fibronectin leucine rich transmembrane protein 3 (FLRT3, and possibly other ligands) to trigger the pathway. We hypothesized that the Tim-3-galectin-9 pathway may be involved in the immune escape of cancer cells of different origins. We found that studied breast tumors expressed significantly higher levels of both galectin-9 and Tim-3 compared to healthy breast tissues of the same patients and that these proteins were co-localized. Increased levels of LPHN2 and expressions of LPHN3 as well as FLRT3 were also detected in breast tumor cells. Activation of this pathway facilitated the translocation of galectin-9 onto the tumor cell surface, however no secretion of galectin-9 by tumor cells was observed. Surface-based galectin-9 was able to protect breast carcinoma cells against cytotoxic T cell-induced death. Furthermore, we found that cell lines from brain, colorectal, kidney, blood/mast cell, liver, prostate, lung, and skin cancers expressed detectable amounts of both Tim-3 and galectin-9 proteins. The majority of cell lines expressed one of the LPHN isoforms and FLRT3. We conclude that the Tim-3-galectin-9 pathway is operated by a wide range of human cancer cells and is possibly involved in prevention of anti-tumor immunity.

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Abstract: The progression of acute myeloid leukemia (AML)—the most severe blood/bone marrow cancer—is determined by the ability of malignant cells to escape host immune surveillance. However, the systemic regulatory mechanisms underlying this phenomenon remain largely unknown. In this study, we discovered a fundamental systemic biochemical strategy that allows AML cells to employ physiological systems within the body to survive and escape immune attack. We found that AML cells use a crucial human adrenal cortex hormone (cortisol) to induce the expression of neuronal receptor latrophilin 1 (LPHN1), which facilitates exocytosis. This receptor interacts with the blood plasma protein fibronectin leucine rich transmembrane protein 3 (FLRT3) to cause secretion of the immune suppressor galectin-9, which impairs the anticancer activities of cytotoxic lymphoid cells. AML is a cancer of the blood and bone marrow that originates from self-renewing malignant immature myeloid cells and rapidly progresses into a systemic, and very often fatal, malignancy.1 AML cells employ physiological systems in the body to produce factors required for proliferation/disease progression.2,3 This includes the hijacking of stem cell factor (SCF), a major hematopoietic growth factor that controls AML progression and thus can become highly oncogenic.2,3 The expression and release of SCF can be triggered by AML cells via cytokines (e.g., interleukin-1β).2 Recent evidence clearly demonstrated that AML cells are also capable of impairing the activities of cytotoxic lymphoid cells (e.g., natural killer (NK) cells and cytotoxic T cells).4 One of the biochemical mechanisms underlying this phenomenon lies in the ability of AML cells to secrete the protein galectin-9. This tandem-type galectin binds the immune receptor Tim-3 and induces a variety of intracellular and cell-to-cell signaling events leading to the inactivation of NK cells, as well as the death of cytotoxic T cells.4,5 We recently reported that the process of galectin-9 secretion in AML cells is stimulated by the unique G protein-coupled receptor LPHN1, which normally functions in neurons to facilitate exocytosis.4,6 LPHN1 is also found in hematopoietic stem cells (HSCs), but its expression disappears at the early stages of their maturation.4,7 However, upon malignant transformation, AML cells preserve their abilities to express LPHN1 and to produce high levels of galectin-9 and Tim-3, in which the latter is involved in trafficking galectin-9 during the secretion process (HSCs express neither galectin-9 nor Tim-34). It is currently unknown which molecular mechanisms trigger elevated levels of LPHN1 expression in primary human AML cells, and in general, the mechanisms of upregulation of LPHN1 expression at the genomic level remain unclear. It is also unknown whether FLRT3, a natural LPHN1 ligand,4,8 is present in human blood plasma and in other tissues associated with AML. Unraveling these mechanisms is crucial to understanding the pathways that control the ability of AML cells to protect themselves against cytotoxic lymphoid cells and, thus, was the aim of the present study.

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Abstract: High mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1β) production by healthy leukocytes, leading to IL-1β-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.