Autocrine VEGF signaling promotes proliferation of neoplastic Barrett's epithelial cells through a PLC-dependent pathway

Abstract

Background & aims: Tumor cells express vascular endothelial growth factor (VEGF), which induces angiogenesis. VEGF also activates VEGF receptors (VEGFRs) on or within tumor cells to promote their proliferation in an autocrine fashion. We studied the mechanisms of autocrine VEGF signaling in Barrett's esophagus cells.

Methods: Using Barrett's epithelial cell lines, we measured VEGF and VEGFR messenger RNA and protein, and studied the effects of VEGF signaling on cell proliferation and VEGF secretion. We studied the effects of inhibiting factors in this pathway on levels of phosphorylated phospholipase Cγ1 (PLCG1), protein kinase C, and extracellular signal-regulated kinases (ERK)1/2. We performed immunohistochemical analysis of phosphorylated VEGFR2 on esophageal adenocarcinoma tissues. We studied effects of sunitinib, a VEGFR2 inhibitor, on proliferation of neoplastic cells and growth of xenograft tumors in mice.

Results: Neoplastic and non-neoplastic Barrett's cells expressed VEGF and VEGFR2 messenger RNA and protein, with higher levels in neoplastic cells. Incubation with recombinant human VEGF significantly increased secretion of VEGF protein and cell number; knockdown of PLCG1 markedly reduced the recombinant human VEGF-stimulated increase in levels of phosphorylated PLCG1 and phosphorylated ERK1/2 in neoplastic cells. Esophageal adenocarcinoma tissues showed immunostaining for phosphorylated VEGFR2. Sunitinib inhibited VEGF signaling in neoplastic cells and reduced weight and volume of xenograft tumors in mice.

Conclusions: Neoplastic and non-neoplastic Barrett's epithelial cells have autocrine VEGF signaling. In neoplastic Barrett's cells, VEGF activation of VEGFR2 initiates a PLCG1-protein kinase C-ERK pathway that promotes proliferation and is self-sustaining (by causing more VEGF production). Strategies to reduce autocrine VEGF signaling (eg, with sunitinib) might be used to prevent or treat cancer in patients with Barrett's esophagus.

Keywords: Apoptosis; BAR-T; BrdU; ERK; Esophageal Cancer; Mouse Model; PKC; PLC; PLCG1; VEGF; VEGF-NA; VEGFR; Vascular Endothelial Growth Factor Receptor 2; bromodeoxyuridine; extracellular signal-regulated kinase; mRNA; messenger RNA; non-neoplastic, telomerase-immortalized Barrett's epithelial cell; phospholipase C; phospholipase Cγ1; protein kinase C; recombinant human vascular endothelial growth factor; rhVEGF; shRNA; short-hairpin RNA; siRNA; small interfering RNA; vascular endothelial growth factor; vascular endothelial growth factor neutralization antibody; vascular endothelial growth factor receptor.

Figure 1

Upregulation of VEGF (A&B) and VEGFR2 (C&D) mRNA and protein in transformed Barrett’s and OE33 adenocarcinoma cells. Data are the means ± SEM of at least 2 separate experiments. *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001; ****, p≤ 0.0001 compared to BAR-T cells.

Figure 2

(A) Representative photomicrographs showing expression of phospho-VEGFR2 in an esophageal adenocarcinoma (right panels) and in histologically-normal esophageal squamous epithelium (left panels) from the same patient. Lower panels show high magnification (400X) of the boxed area in the upper panels (100X). (B) Phospho-VEGFR2 expression in fresh tissue biopsies from two patients. (C&D) By 60 minutes, treatment with rhVEGF causes nuclear translocation of phospho-VEGFR2 in transformed Barrett’s (P13R1) and OE33 cells containing control siRNA, but not VEGFR2 siRNA as detected by (B) Western blot and (C) immunofluorescence. VEGFR2 was stained with anti-human phospho-VEGFR2 antibody (turquoise) and nuclei were counterstained with DAPI (blue).

Figure 2

(A) Representative photomicrographs showing expression of phospho-VEGFR2 in an esophageal adenocarcinoma (right panels) and in histologically-normal esophageal squamous epithelium (left panels) from the same patient. Lower panels show high magnification (400X) of the boxed area in the upper panels (100X). (B) Phospho-VEGFR2 expression in fresh tissue biopsies from two patients. (C&D) By 60 minutes, treatment with rhVEGF causes nuclear translocation of phospho-VEGFR2 in transformed Barrett’s (P13R1) and OE33 cells containing control siRNA, but not VEGFR2 siRNA as detected by (B) Western blot and (C) immunofluorescence. VEGFR2 was stained with anti-human phospho-VEGFR2 antibody (turquoise) and nuclei were counterstained with DAPI (blue).

Figure 2

(A) Representative photomicrographs showing expression of phospho-VEGFR2 in an esophageal adenocarcinoma (right panels) and in histologically-normal esophageal squamous epithelium (left panels) from the same patient. Lower panels show high magnification (400X) of the boxed area in the upper panels (100X). (B) Phospho-VEGFR2 expression in fresh tissue biopsies from two patients. (C&D) By 60 minutes, treatment with rhVEGF causes nuclear translocation of phospho-VEGFR2 in transformed Barrett’s (P13R1) and OE33 cells containing control siRNA, but not VEGFR2 siRNA as detected by (B) Western blot and (C) immunofluorescence. VEGFR2 was stained with anti-human phospho-VEGFR2 antibody (turquoise) and nuclei were counterstained with DAPI (blue).

Figure 3

Treatment with (A) VEGF neutralization antibody (VEGF-NA), (B) SU1498, and (D) VEGFR2 siRNA causes a reduction in BrdU incorporation in transformed Barrett’s and OE33 cells. (E) VEGFR2 siRNA decreases VEGF secretion by the neoplastic cells. Data are means ± SEM of 3 separate experiments. *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001 compared to corresponding controls. (C) Representative Western blot demonstrating VEGFR2 knockdown by siRNA.

Figure 4

Treatment with rhVEGF causes an increase in (A) VEGF secretion and (B) cell number in transformed Barrett’s and OE33 cells. Data are the means ± SEM of 3 separate experiments. *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001 compared to corresponding controls. (C) Representative Western blots demonstrating phosphorylation of VEGFR2, PLC-γ1, PKC-α/β, and ERK1/2, and Akt by rhVEGF in the neoplastic cells. Numbers represent the relative quantity of protein with respect to the loading control.

Figure 5

Representative Western blots demonstrating that siRNAs against VEGFR2 and PLC-γ decrease rhVEGF-induced phospho-PLC-γ1, PKC-α/β, and ERK1/2 in (A) transformed Barrett’s and (B) OE33 cells. PLC-γ siRNA decreases (C) VEGF secretion and (D) BrdU incorporation in transformed Barrett’s and OE33 cells. Data are the means ± SEM of 3 separate experiments. *, p≤ 0.05; **, p≤ 0.01 compared to corresponding controls. (E) Mouse xenografts of transformed, parental P13R1 cells (without VEGFR2 shRNA) exhibited robust tumor growth, whereas both the population and selected clone (B3) of P13R1 cells with VEGFR2 knockdown exhibited no tumor growth whatsoever.

Figure 5

Representative Western blots demonstrating that siRNAs against VEGFR2 and PLC-γ decrease rhVEGF-induced phospho-PLC-γ1, PKC-α/β, and ERK1/2 in (A) transformed Barrett’s and (B) OE33 cells. PLC-γ siRNA decreases (C) VEGF secretion and (D) BrdU incorporation in transformed Barrett’s and OE33 cells. Data are the means ± SEM of 3 separate experiments. *, p≤ 0.05; **, p≤ 0.01 compared to corresponding controls. (E) Mouse xenografts of transformed, parental P13R1 cells (without VEGFR2 shRNA) exhibited robust tumor growth, whereas both the population and selected clone (B3) of P13R1 cells with VEGFR2 knockdown exhibited no tumor growth whatsoever.

Figure 6

Sunitinib decreases (A) phospho-PLC-γ1 protein expression, (B) VEGF secretion, (C) cell number and (D) BrdU incorporation in a dose dependent manner in transformed Barrett’s and OE33 cells. Data are the means ± SEM of at least 3 separate experiments. **, p≤ 0.01; ***, p≤ 0.001 compared to corresponding controls; +, p≤ 0.05; ++, p≤ 0.01; +++, p≤ 0.001 compared to corresponding 5 μM treated cells.

Figure 7

In mouse xenografts of transformed Barrett’s cells, sunitinib (A) delays index tumor growth, (B) decreases growth rate per day in index tumor volume, and (C) decreases index tumor volume at sacrifice. Sunitinib-treated tumors have (D) lower total tumor weights, (E) lower numbers of Ki-67 positive staining cells and no difference in microvessel density compared to vehicle-treated tumors. *, p≤ 0.05 compared to vehicle. (F) Schematic model demonstrating the pro-proliferative effect of autocrine VEGF/VEGFR2 signaling and sunitinib’s inhibition of this pathway in neoplastic Barrett’s cells.

Figure 7

In mouse xenografts of transformed Barrett’s cells, sunitinib (A) delays index tumor growth, (B) decreases growth rate per day in index tumor volume, and (C) decreases index tumor volume at sacrifice. Sunitinib-treated tumors have (D) lower total tumor weights, (E) lower numbers of Ki-67 positive staining cells and no difference in microvessel density compared to vehicle-treated tumors. *, p≤ 0.05 compared to vehicle. (F) Schematic model demonstrating the pro-proliferative effect of autocrine VEGF/VEGFR2 signaling and sunitinib’s inhibition of this pathway in neoplastic Barrett’s cells.