Regulatory role of DEPTOR-mediated cellular autophagy and mitochondrial reactive oxygen species in angiogenesis in multiple myeloma

Abstract

DEPTOR, an inhibitor of mammalian target of rapamycin (mTOR), is essential for the survival of multiple myeloma (MM) cells. The expression level of dEPTOR is closely related to the prognosis of patients with MM treated with the antiangiogenic agent thalidomide; however, its role in the regulation of angiogenesis has not yet been elucidated. In the present study, the expression levels of dEPTOR and vascular endothelial growth factor (VEGF), and the microvessel density (MVd) of bone marrow (BM) from patients with MM assessed. dEPTORoverexpression plasmid or cRISPR-associated protein 9 (cas9) and single guided RNAs (sgRNAs) were used to modulate dEPTOR expression. The dEPTOR-mediated angiogenic effects were assessed using a tube formation assay of human umbilical vein endothelial cells (HUVEcs) cultured in the collected conditioned medium from MM cell lines with different expression levels of dEPTOR. It was found that the expression level of dEPTOR negatively correlated with the VEGF level and BM MVd in MM. Autophagic activity was regulated by dEPTOR expression, but was not related to thalidomide-binding protein cRBN, which is required for thalidomide to play an anti-tumor and antiangiogenic role in MM cells. The disruption of dEPTOR protein decreased cellular autophagy, increased VEGF expression in MM cells, and inhibited the tube formation of HUVEcs, while a high expression of dEPTOR exerted the opposite effect. Moreover, targeting dEPTOR also resulted in the production of mitochondrial reactive oxygen species (mtROS), the phosphorylation of nuclear factor-κB (NF-κB) and an increase in interleukin 6 (IL-6) secretion. Of note, these effects are fully abrogated by treatment with autophagy activator (SMER28) or mitochondrial-specific antioxidant (Mito-TEMPO). Taken together, the present study demonstrates the role of dEPTOR in the regulation of autophagy/mtROS and subsequent angiogenesis. The results provide a novel mechanism for the further understanding of the therapeutic effects of thalidomide on MM.