Tumor blood vessels are formed by two different mechanisms, namely tumor angiogenesis and vasculogenesis, mostly through the vascular endothelial growth factor (VEGF)/VEGF receptor-2 (VEGFR-2) axis (1). Tumor angiogenesis occurs through the proliferation and migration of endothelial cells from pre-existing neighboring blood vessels, whereas tumor vasculogenesis is initiated by recruiting bone marrow-derived VEGFR-2+ endothelial progenitor cells (EPCs), which differentiate into mature endothelial cells (2). Thus, VEGF released from the tumor microenvironment plays an important role in the formation of tumor blood vessels, leading to tumor progression and metastasis. It is now well understood that blocking the VEGF/VEGFR-2 pathway can act as a therapeutic strategy for cancer patients by inhibiting the functions of endothelial cells and EPCs (3, 4).
Low-dose metronomic chemotherapy (LDMC) has recently been attracting attention as a promising alternative to conventional maximum-tolerated dose chemotherapy (5). This regimen is thought to effectively target both activated or proliferating tumor endothelial cells as well as EPCs, which are more sensitive to low-dose anticancer drugs than normal endothelial and tumor cells (3, 4). Indeed, LDMC has been shown to effectively inhibit the tumor progression by suppressing tumor angiogenesis through functional impairment of tumor endothelial cells and bone marrow mononuclear cell (BMMNC)-derived EPCs (4, 5). This is affected by a systemic increase in anti-angiogenic factors, including thrombospondin-1 (TSP-1), as well as a reduction in pro-angiogenic factors, such as VEGF and VEGFR-2 (3, 6). However, the molecular mechanism underlying anti-angiogenesis by LDMC has not been clearly elucidated.
The protein regulated in development and DNA damage response 1 (REDD1) is upregulated upon the treatment with chemotherapeutic drugs, including doxorubicin (DOX) (7, 8). REDD1 functions as an endogenous mTORC1 inhibitor as well as an atypical NF-κB activator (9, 10). The mTORC1 pathway plays a crucial role in the regulation of various cellular functions, such as protein biosynthesis and angiogenesis (11). Indeed, the mTOR inhibitor rapamycin suppresses tumor angiogenesis by downregulating VEGF/VEGFR expression through the inhibition of translational initiation (12, 13). We have recently reported that LDMC-induced REDD1 suppresses tumor angiogenesis and tumor progression via functional impairment of tumor endothelial cells through translational repression of
Here, we hypothesized that REDD1 is responsible for the LDMC-mediated impairment of EPC function and tumor angiogenesis. We found that LDMC with DOX increased REDD1 expression, repressed
Since LDMC induces REDD1 expression and impairs endothelial cell functions (3, 6-8), we examined whether low-dose DOX treatment-induced REDD1 regulates angiogenic receptors in BMMNC-derived EPCs. When treated with non-cytotoxic doses (1-3 nM) of DOX as determined by assaying cytotoxicity and cell viability (Supplementary Fig. 1A, B), BMMNC-derived EPCs showed dose-dependently increased REDD1 expression, along with downregulation of VEGFR-2; however, there were no differences in the expression levels of VEGFR-1, epithermal growth factor receptor (EGFR), and insulin growth factor-1 receptor β (IGF-1Rβ) (Fig. 1A). Since REDD1 is known as an endogenous mTORC1 inhibitor (9), we next compared the effects of DOX, REDD1, and the mTORC1 inhibitor rapamycin on VEGFR-2 expression. Treatment with DOX or rapamycin or adenoviral overexpression of REDD1 suppressed VEGFR-2 expression in BMMNC-derived EPCs, without affecting its mRNA levels, compared with untreated control cells (Fig. 1B, C), suggesting that DOX selectively inhibits the translational expression of VEGFR-2 by REDD1-dependent mTORC1 inhibition. We further examined the possible role of REDD1 in DOX-mediated VEGFR-2 downregulation by analyzing the mTORC1 signaling pathway and polysome profiling. DOX treatment or REDD1 overexpression inhibited the phosphorylation of mTOR, p70 ribosomal S6 kinase (S6K), and eukaryotic translational initiation factor 4E-binding protein 1 (4E-BP1) (Fig. 1D). Consistent with this, both treatments decreased the assembly of high-molecular-weight polysome complexes and polysome-associated
Since VEGFR-2 plays a crucial role in EPC mobilization and differentiation (14, 15), we examined the effects of DOX treatment and REDD1 overexpression on EPC differentiation from cultured BMMNCs isolated from WT and
Since the VEGF/VEGFR-2 system regulates the endothelial nitric oxide synthase (eNOS)/NO axis, which is another functional marker of EPCs (16), we investigated whether low-dose DOX regulates eNOS-dependent NO production in BMMNC-derived EPCs. VEGF-A stimulation significantly increased NO production in EPCs normally differentiated from both WT and
Since LDMC inhibits tumor angiogenesis by impairing the mobilization and recruitment of EPCs as well as pre-existing endothelial cell functions (3, 17, 18), we investigated whether metronomic DOX treatment regulates the mobilization of EPCs from the bone marrow into the peripheral blood in B16 melanoma-bearing WT and
We investigated the functional role and molecular mechanism of REDD1, a cellular mTORC1 inhibitor, in the inhibitory effects of low-dose DOX treatment on the differentiation and mobilization of EPCs. Our data showed that low-dose DOX treatment inhibited the differentiation of BMMNCs into EPCs by upregulating REDD1 and subsequently repressing mTORC1-dependent
Although angiogenic processes are well understood, it has been shown that several single antiangiogenic therapies targeting tumor endothelial cells do not always effectively inhibit tumor angiogenesis and tumor progression (19), suggesting that tumor angiogenesis is not regulated only by the stimulation of pre-existing endothelial cells. Asahara and his colleagues have identified a population of circulating EPCs with characteristics similar to those of embryonic angioblasts that contribute to angiogenesis in ischemic tissues and tumors (2, 20, 21). In general, EPCs promote tumor angiogenesis in three consecutive steps, namely mobilization from bone marrow, recruitment to the tumor site, and differentiation into mature endothelial cells; all of these processes can be regulated by the signaling pathways induced by growth factors or chemokines and their corresponding receptors, including VEGF-A/VEGFR-2, stromal cell-derived factor-1 (SDF-1)/CXCR4, and basic fibroblast growth factor/its receptor (3). Therefore, treatment with blockades of the VEGF-A/VEGFR-2 and SDF-1/CXCL4 axes has been shown to decrease the number of circulating EPCs and their recruitment into tumor tissues in patients with rectal cancer and Lewis lung cancer mouse models, respectively, resulting in amelioration of tumor angiogenesis and tumor progression (22, 23). LDMC has been shown to reduce the number of circulating EPCs in tumor-bearing mice and cancer patients, leading to a parallel decrease in tumor angiogenesis and vessel density (3); however, the mechanism by which LDMC inhibits EPC-mediated tumor angiogenesis remains unclear. Our results demonstrate that low-dose DOX treatment downregulates VEGFR-2 expression along with the suppression of differentiation, mobilization, NO production, and angiogenic activity of EPCs in WT but not
REDD1 is required for the dissociation of TSC2/14-3-3 and subsequent inhibition of the mTORC1 pathway, which is essential for controlling cap-dependent mRNA translation (9, 24), and is induced by various stress conditions and chemotherapeutic drugs, including DOX (3, 7, 9). Consistent with this, we found that DOX upregulated REDD1 and subsequently impeded the assembly of high-molecular-weight-polysome complexes by inhibiting the mTORC1-dependent phosphorylation of S6K and 4E-BP1. Notably, DOX treatment caused translational repression of
In conclusion, our results demonstrate that low-dose metronomic DOX treatment inhibited the differentiation and mobilization of EPCs from bone marrow through selective translational repression of
Additional materials and methods are available in the Supplemental Information.
Bone marrow was collected from the tibia and femur of 6-week-old male mice in a 50 ml conical tube immediately after sacrifice by CO2 inhalation. BMMNCs were isolated by density gradient centrifugation using Histopaque-1083 (Sigma-Aldrich), suspended in EGM-2MV (Lonza, #CC-3202), and seeded on gelatin-coated plates at a density of 1 × 106 cells/cm2. Four days later, non-adherent cells were washed off with phosphate-buffered saline (PBS), cultured in fresh media for 2 days, and then treated daily with DOX (3 nM) or rapamycin (20 nM) in fresh media for 24 h. In addition, adherent BMMNCs were infected with adenovirus expressing mouse
Cells were lysed in 400 μl lysis buffer [15 mM Tris-HCl (pH 7.4), 0.3 M NaCl, 15 mM MgCl2, 0.1 mg/ml cycloheximide, and 200 U/ml Superase-InTM (Ambion, Waltham, MA, USA)]. After centrifugation at 2000 ×
BMMNCs treated with drugs or infected with Ad-
Statistical analyses were performed using GraphPad Prism software (version 6.07). All values are presented as mean ± standard error of mean (SEM). Statistical significance was determined using two-tailed Student’s t-test for comparison between two groups or two-way ANOVA with Holm-Sidak’s multiple comparisons test for comparison between multiple groups. Statistical significance was set at P < 0.05.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2017R1A2B3004565).
The authors have no conflicting interests.