Mesencephalic astrocyte-derived neurotrophic factor, a potential immunomodulator, is secreted from interferon-γ –stimulated tumor cells through ER calcium depletion

Introduction: The most successful immunotherapeutic agents are blocking antibodies to either programmed cell death-1 (PD-1), an inhibitory receptor expressed on T lymphocytes, or to its ligand, programmed cell death-ligand 1 (PD-L1). Nevertheless, many patients do not respond, and additional approaches, specifically blocking other inhibitory receptors on T cells, are being explored. Importantly, the ligands for these receptors are often expressed on the surface of the tumor cells. Indeed, cancer cells express high levels of PD-L1 upon stimulation with interferon-γ (IFN-γ), a major cytokine in the tumor microenvironment. The increase in PD-L1 expression serves as a negative feedback towards the immune system, and allows the tumor to evade the attack of immune cells. We hypothesized that additional immunomodulator ligands are present on cancer cells, and similar to PD-L1, their expression is increased in the inflammatory microenvironment. Methods: A mass spectrometry-based screen was performed, in which tumor cells were treated with IFN-γ and their membrane associated proteome was analyzed. Results: 60 membrane-associated proteins were upregulated upon IFN-γ treatment, of which 17 are known drug targets, while 16 have never been implicated therapeutically in cancer and therefore are possible drug targets. To validate the mass spectrometry results, we focused on one of the proteins that have not been implicated directly in cancer, mesencephalic astrocyte-derived neurotrophic factor (MANF). MANF is known to be secreted upon ER calcium depletion, to bind phospholipids on membranes, and to induce an alternatively activated macrophage phenotype (M2), which may support tumor growth. We found that both membrane-bound and secreted MANF were increased in IFN-γ-treated cells, demonstrated by western blot analysis and ELISA. IFN-γ induced MANF secretion from diverse cell-lines - melanoma cells, lung cancer cells, colon carcinoma cells and hepatoma cells. Surprisingly, there was no increase in MANF RNA or intracellular protein level upon IFN-γ stimulation. However, IFN-γ induced ER calcium depletion, which can explain MANF secretion. Indeed, Dantrolene, an inhibitor of ER calcium release, prevented MANF secretion. Conclusion: MANF is secreted from IFN-γ-stimulated tumor cells; thus, it may serve as an immunomodulator and further studies will be conducted to elucidate its role in tumor immunity.