Oncometabolite succinate promotes angiogenesis by upregulating VEGF expression through GPR91-mediated STAT3 and ERK activation

Abstract

Altered cellular metabolism is now generally acknowledged as a hallmark of cancer cells, the resultant abnormal oncometabolites cause both metabolic and nonmetabolic dysregulation and potential transformation to malignancy. A subset of cancers have been found to be associated with mutations in succinate dehydrogenase genes which result in the accumulation of succinate. However, the function of succinate in tumorigenesis remains unclear. In the present study, we aim to investigate the role of oncometabolite succinate in tumor angiogenesis. Our data demonstrated the accumulation of markedly elevated succinate in gastric cancer tissues compared with that in paracancerous tissues. Moreover, succinate was able to increase the chemotactic motility, tube-like structure formation and proliferation of primary human umbilical vascular endothelial cells (pHUVECs) in vitro, as well as promoting the blood vessel formation in transgenic zebrafish. Our mechanistic studies reveal that succinate upregulates vascular endothelial growth factor (VEGF) expression by activation of signal transducer and activator of transcription 3 (STAT3) and extracellular regulated kinase (ERK)1/2 via its receptor GPR91 in a HIF-1α independent mechanism. Taken together, these data indicate an important role of the succinate-GPR91 axis in tumor angiogenesis, which may enable development of a novel therapeutic strategy that targets cancer metabolism.

Keywords: ERK1/2; GPR91; angiogenesis; gastric cancer; succinate.

Figure 1. Succinate level is significantly higher in human gastric cancer tissues and cancer cell lines compared with adjacent tissues and normal cell line, respectively

A. Succinate levels were measured in twelve pairs of human gastric adenocarcinoma and tumor-adjacent tissues, which were collected from stage II-IV gastric cancer patients. **p<0.01 compared with adjacent tissues. B. Succinate levels were determined in the culture supernatants of human gastric cancer cells and normal gastric epithelial cells. *p<0.05 and **p<0.01 versus the culture supernatants of GES-1 cells.

Figure 2. Succinate increases the proliferation, chemotactic motility and capillary structure formation of pHUVECs in vitro

A. BrdU incorporation assay was used to determine the proliferation of pHUVECs in the presence of various concentration of succinate. B. Wound-healing migration assay was applied to observe the effect of succinate on pHUVECs migration. The confluent cells were scratched by pipette and cultured in the presence of various concentration of succinate (magnification, ×100). C. Effect of succinate on pHUVECs migration was also evaluated by transwell assay. pHUVECs were seeded in the upper transwell chamber. The bottom chamber was filled with various concentrations of succinate. After 8 h of co-culture, the migrated cells were quantified by manual counting (Magnification, ×100). D. Capillary-like formation of pHUVECs was observed in the presence of various concentration of succinate. pHUVECs (2.0 ×10⁴ cells/well) were placed in 96-well plates coated with growth factor reduced matrigel. Six hours later, tubular structures were photographed (Magnification, ×100). Three independent experiments (A, B, C and D) data are presented as mean ±SD. *p<0.05 and **p<0.01 versus PBS control.

Figure 3. Succinate promotes vessel formation in the zebrafish embryo

A. Effect of succinate on angiogenesis was evaluated in zebrafish embryos. Transgenic (flk1: GFP) zebrafish embryos were picked out and distributed into a 24-well microplate (9 embryos/well), and pre-treated with 0.2 μg/mL PTK787 (VEGFR inhibitor, Sigma). Then, various concentration of succinate (0, 100, 200, 400, 800 μM) or 10 ng/mL of VEGF₁₆₅ were added to the culture for twenty-four hours. Representative photographs are listed to show the subintestinal vessels (SIVs) indicated by white arrow. B, C. Quantitative analysis of the overall length of zebrafish SIVs are shown. Data are presented as mean ±SD.*p<0.05 and **p<0.01 versus PBS control.

Figure 4. Succinate activates ERK1/2 and STAT3 signaling through GPR91 in pHUVECs

A. STAT3 and ERK1/2 activation were measured by Western blotting in pHUVECs treated with 400μM succinate for different periods of time. B. STAT3 and ERK1/2 activation were determined in pHUVECs cultured in the presence of various concentrations of succinate or 10 ng/mL of VEGF₁₆₅ for 15 min. C. STAT3 and ERK1/2 activation were evaluated in pHUVECs transduced with LV.shScrambled or LV. shGPR91-1, or LV. shGPR91-2 in the presence of succinate for 15 min. The expression of total STAT3 and Erk1/2 protein were also detected. β-actin was used as loading control. Data were represented as mean ± SD; n = 3. *P<0.05 versus PBS control.

Figure 5. Succinate promotes angiogenesis by activating ERK1/2 signaling through GPR91

pHUVECs, transduced with LV.shGPR91 or LV.shScrambled, were pre-treated with 10μM ERK1/2 inhibitor selumetinib and then cultured in the presence of succinate to evaluate its pro-angiogenic effect. A. BrdU incorporation assay was used to determine the effect of succinate on the proliferation of pHUVECs. B. The wound-healing assay was applied to observe the effect of succinate on pHUVECs migration (magnification, ×100). C. Transwell assay was also adopted to determine the effect of succinate on pHUVECs migration (Magnification, ×100). D. Capillary-like formation of pHUVECs was observed in the presence of succinate (Magnification, ×100). The data from three independent experiments are presented as mean±SD. *p<0.05 and **p<0.01 versus LV.shScrambled group.

Figure 6. Succinate enhances VEGF expression by activating ERK1/2 and STAT3 signaling through GPR91

A. Succinate increases VEGF expression independent of HIF-1α gene. The expression of HIF-1α protein in LV.shScrambled-transduced or LV.shHIF-1α-transduced AGS cells was determined by Western blotting (left). The cell culture supernatant was collected from the transduced cells in the presence of 400 μM succinate for 24h and measured VEGF level (right). VEGF mRNA levels B. or protein concentration in the supernatant C. were determined by RT-qPCR or ELISA in pHUVECs cultured in the presence of succinate for 12h, which were pre-treated with 10μM Selumetinib (ERK1/2 inhibitor) or/and 10μM Stattic (STAT3 inhibitor) for ten minutes. Each column represents the mean±SD (n=3). *P<0.05 versus PBS control.