Autocrine VEGF signaling promotes cell proliferation through a PLC-dependent pathway and modulates Apatinib treatment efficacy in gastric cancer

Yi Lin, Ertao Zhai, Bing Liao, Lixia Xu, Xinhua Zhang, Sui Peng, Yulong He, Shirong Cai, Zhirong Zeng, Minhu Chen, Yi Lin, Ertao Zhai, Bing Liao, Lixia Xu, Xinhua Zhang, Sui Peng, Yulong He, Shirong Cai, Zhirong Zeng, Minhu Chen

Abstract

Background: Tumor cells produce vascular endothelial growth factor (VEGF) which interact with the membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth in an angiogenesis-independent fashion. Apatinib, a highly selective VEGFR2 inhibitor, is the only effective drug for patients with terminal gastric cancer (GC) who have no other chemotherapeutic options. However, its treatment efficacy is still controversy and the mechanism behind remains undetermined. In this study, we aimed to investigate the role of autocrine VEGF signaling in the growth of gastric cancer cells and the efficacy of Apatinib treatment.

Methods: The expression of phosphor VEGFR2 in gastric cancer cell lines was determined by real-time PCR, immunofluorescence, and Western blot. The gastric cancer cells were administrated with or without recombination human VEGF (rhVEGF), VEGFR2 neutralizing antibody, U73122, SU1498, and Apatinib. The nude mice were used for xenograft tumor model.

Results: we found that autocrine VEGF induced high VEGFR2-expression, promoted phosphorylation of VEGFR2, and further enhanced internalization of pVEGFR2 in gastric cancer cells. The autocrine VEGF was self-sustained through increasing VEGF mRNA and protein expression. It exerted pro-proliferative effect through a PLC-ERK1/2 dependent pathway. Furthermore, we demonstrated that in VEGFR2 overexpressing gastric cancer cells, Apatinib inhibited cell proliferation in vitro and delayed xenograft tumor growth in vivo. However, these effects were not observed in VEGFR2 low expressing gastric cancer cells.

Conclusion: These results suggested that autocrine VEGF signaling promotes gastric cancer cell proliferation and enhances Apatinib treatment outcome in VEGFR2 overexpression gastric cancer cells both in vitro and in vivo. This study would enable better stratification of gastric cancer patients for clinical treatment decision.

Keywords: Apatinib; VEGF; autocrine; gastric cancer; proliferation.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declared that they have no competing interests.

Figures

Figure 1. Differential Expression of VEGF, pVEGFR2,…
Figure 1. Differential Expression of VEGF, pVEGFR2, and VEGFR2 in gastric cancer cell lines
A. Expression of VEGF, VEGFR2 was analyzed by qRT-PCR in 5 gastric cancer cell lines. B. Expression VEGFR2, pVEGFR2, VEGF protein was analyzed by Western blot in 5 gastric cancer cell lines. C. ELISA analysis of the secretion of VEGF in gastric cancer cell lines. D. Expression of VEGFR2, pVEGFR2, and VEGF in the cell membrane, cytoplasm, and nucleus of SGC-7901, BGC-823, and MGC-803 cell lines. E. Expression of VEGFR2 and pVEGFR2 was analyzed by IF in SGC-7901 and BGC-823 cells.
Figure 2. Inhibition of VEGF-VEGFR2 signaling decreased…
Figure 2. Inhibition of VEGF-VEGFR2 signaling decreased cell proliferation and VEGF secretion in gastric cancer cell lines
A. Proliferation of gastric cancer cells in response to VEGF-neutralization antibodies (VEGF-NA) in SGC-7901, BGC-823, and MGC-803 cells. B. The proliferation of gastric cancer cells in response to VEGF receptor 2 neutralization antibodies (VEGFR2-NA) in SGC-7901, BGC-823, and MGC-803 cells. C. The proliferation of gastric cancer cells in response to SU1498 in SGC-7901, BGC-823 and MGC-803 cells. D. VEGF secretion of gastric cancer cells in response to VEGF-neutralization antibodies (VEGF-NA) in SGC-7901, BGC-823, and MGC-803 cells. E. VEGF secretion of gastric cancer cells in response to VEGF receptor 2 neutralization antibodies (VEGFR2-NA) in SGC-7901, BGC-823, and MGC-803 cells. F. VEGF secretion of gastric cancer cells in response to SU1498 in SGC-7901, BGC-823 and MGC-803 cells. Mean±SEM, t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 3. Exogenous VEGF promoted pVEGFR2 nuclear…
Figure 3. Exogenous VEGF promoted pVEGFR2 nuclear translocation
A. Expression of pVEGFR2 and VEGF after treating cells with rhVEGF in total protein (left panel) and in nuclear protein (right panel). B. Blocking VEGFR2 by SU1498, the expression of pVEGFR2 and VEGF in nuclear protein. C. Blocking VEGFR2 by SU1498, the expression of pVEGFR2 were measured by IF.
Figure 4. Autocrine VEGF signaling promoted cell…
Figure 4. Autocrine VEGF signaling promoted cell proliferation and its own production in gastric cancer cells
A. The viability of gastric cancer cells in response to recombinant human VEGF (rhVEGF) in SGC-7901 (left panel), BGC-823 (middle panel), and MGC-803 cells (right panel). B. Proliferation of gastric cancer cells under basal medium (BM) and condition medium (CM) with or without VEGF-NA in SGC-7901 (left panel), BGC-823 (middle panel), and MGC-803 cells (right panel). C. Autocrine VEGF signaling affected gastric cancer cells self-sustained protein level. D. Autocrine VEGF signaling affected gastric cancer cells self-sustained mRNA level. E. Autocrine VEGF signaling promoted self-secretion in SGC-7901 and BGC-823 cells. Mean±SEM, t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 5. Autocrine VEGF signaling promoted cell…
Figure 5. Autocrine VEGF signaling promoted cell proliferation through a VEGFR2-PLCγ1-ERK1/2 pathway in GC
A. gastric cancer cells were treated with rhVEGF and were harvested at different time points. The time course changes of the phosphorylation of VEGFR2, PLC, and ERK1/2 were detected by Western blot. B. The proliferation of gastric cancer cells in response to PLCγ1 inhibitor (U73122) in SGC-7901 and BGC-823 cells. C. Blocking PLCγ1 with U73122, proliferation of gastric cancer cells in response to rhVEGF in in SGC-7901 and BGC-823 cells. D. After treating cells with U73122, the protein levels were measured by Western blot. GAPDH was included as a loading control. Mean±SEM, t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 6. Inhibition of VEGFR2 by Apatinib…
Figure 6. Inhibition of VEGFR2 by Apatinib decreased cell proliferation by blocking VEGFR2-PLCγ1-ERK1/2 pathway and reduced VEGF secretion
A. The viability of gastric cancer cells in response to Apatinib in SGC-7901 (left panel), BGC-823 (middle panel), and MGC-803 cells (right panel). B. Treating with Apatinib, proliferation of gastric cancer cell lines in response to rhVEGF in SGC-7901 (left panel), BGC-823 (middle panel), and MGC-803 cells (right panel). C. Treating cells with Apatinib at different concentration affected secretion of VEGF in SGC-7901 (left panel), BGC-823 (middle panel), and MGC-803 cells (right panel). D. The protein levels were measured by Western blot after treating cells with Apatinib. GAPDH was included as a loading control. Mean±SEM, t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 7. The efficacy of Apatinib on…
Figure 7. The efficacy of Apatinib on suppressing GC growth in xenograft tumor models
A. In nude mouse xenografts of gastric cancer cells that overexpressed VEGFR2 and VEGF, Apatinib delays tumor growth. B. Apatinib decreased tumor volume in SGC-7901 and BGC-823 tumor models, but not in the MGC-803 model when mice were sacrificed. C. Apatinib-treated tumors decreased total tumor weights in SGC-7901 and BGC-823 tumor models, but not in the MGC-803 tumor model. D. Measured by IHC, Apatinib decreased the Ki67 positive rate of tumor cells in SGC-7901 and BGC-823 tumor models, but not in the MGC-803 tumor model. Mean±SEM, t-test, *P<0.05, **P<0.01, ***P<0.001.

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