Angiotensin II type I receptor (AT1R) is an independent prognosticator of esophageal squamous cell carcinoma and promotes cells proliferation via mTOR activation

Abstract

Background: The aim of this study was to investigate the effects of the angiotensin II/ angiotensin II type I receptor (AT1R) and angiotensin II type II receptor (AT2R) signaling pathway in esophageal squamous cell carcinoma (ESCC).

Methods: Immunohistochemistry was performed to evaluate the expression levels of AT1R and AT2R in tissues from 152 surgically resected ESCC patients, and those expression levels were then correlated with treatment outcomes. The angiotensin II/AT1R/AT2R signaling pathway and its biological effects in the context of ESCC were investigated in vitro and in vivo.

Results: In human samples, AT1R overexpression was univariately associated with inferior overall survival and remained multivariately independent (hazard ratio=1.812). In vitro, angiotensin II stimulated the growth of ESCC cells in a dose-dependent manner. Treatment with irbesartan or AT1R-RNAi knockdown but not treatment with PD123319 significantly decreased the level of angiotensin II-induced ESCC cell proliferation. Angiotensin II also caused mTOR activation in a dose-dependent manner, and everolimus or mTOR-RNAi knockdown significantly suppressed the level of angiotensin II-induced ESCC cell proliferation. Furthermore, AT1R-RNAi knockdown suppressed the activation of mTOR. Clinically, AT1R expression was also correlated with phosphorylated mTOR expression. In a xenograft model, local angiotensin II injection enhanced tumor growth, and this effect could be decreased by treatment with irbesartan or everolimus. In a 4-NQO-induced-ESCC murine model, irbesartan significantly decreased the incidence of esophageal tumor.

Conclusions: These findings suggest that AT1R overexpression is an independent adverse prognosticator for patients with ESCC and that angiotensin II/AT1R signaling stimulates ESCC growth, in part through mTOR activation.

Keywords: AT1R; angiotensin II; esophageal cancer; mTOR; squamous cell carcinoma.

Figure 1. Immunohistochemical staining of angiotensin II type I receptor (AT1R), angiotensin II type II receptor (AT2R), and phosphorylated mammalian target of rapamycin (p-mTOR)

A, B. AT1R immunoreactivity was present in normal adrenal gland tissues used as a positive control. C, D. Low expression of AT1R. E, F. Overexpression of AT1R. G, H. AT2R immunoreactivity was present in normal adrenal gland tissues used as a positive control. I, J. Low expression of AT2R. K, L. Overexpression of AT2R. M, N. p-mTOR immunoreactivity was present in normal submucosal glands from the esophagus used as a positive control. O, P. Low expression of p-mTOR. Q, R. Overexpression of p-mTOR.

Figure 2. Kaplan–Meier curves according to angiotensin II type I receptor (AT1R) status. A. Overall survival according to AT1R status

B. Disease-free survival according to AT1R status.

Figure 3. Angiotensin II induced cell proliferation of ESCC was required for AT1R

A. Serum-starved CE81T/VGH, CE48T/VGH, and CE146T/VGH cells treated with or without angiotensin II stimulation were seeded into 96-well plates with 1.0% of FBS. The cells were cultured for 72 hours followed by MTT assay (OD570) to quantitate cell growth. The data were normalized against the OD570 value on control group (0 μM) of each treatment. B. Serum-starved cells were pre-treated with or without various concentrations of irbesartan or losartan or PD123319 for 30 mins; the cells were then stimulated with angiotensin II (10 μM). The cells were cultured for 72 hours followed by MTT assay to quantitate cell growth. C. The mRNA and protein expression profiles of AT1R and AT2R in ESCC cell lines were determined by Q-RT-PCR and Western blotting. D and E. The abilities of cell growth, colony formation, and BrdU incorporation in AT1R-depleted CE81T/VGH cells or siControl group with or without angiotensin II (10 μM) stimulation were assayed. F. The abilities of colony formation and BrdU incorporation in angiotensin II-stimulated CE81T/VGH cells treated with or without irbesartan were assayed.

Figure 4. mTOR expression and activation participated in angiotensin II/AT1R signaling in ESCC

(A, left panel) The AKT activation was examined in AT1R-depleted cells with or without angiotensin II stimulation. (A, right panel) the phosphorylated status of mTOR was determined in ESCC cells stimulated with angiotensin II by Western blotting. B. Serum-starved cells were pre-treated with indicated concentrations of everolimus for 30 mins; the cells were then stimulated with or without angiotensin II. The cells were cultured for 72 hours followed by MTT assay to quantitate cell growth. In addition, the abilities of colony formation and BrdU incorporation in angiotensin II-stimulated CE81T/VGH cells treated with or without everolimus were assayed. C. The protein expression levels of total mTOR, phosphorylated mTOR and AT1R were demonstrated in CE81T/VGH cells transfected with siControl and simTOR. The cell growth abilities of siControl and simTOR stimulated with angiotensin II were measured by MTT assay. D. The protein expression profiles of AT1R, total mTOR, and phosphorylated mTOR were determined in AT1R-depleted CE81T/VGH cells.

Figure 5. Angiotensin II injection enhanced ESCC xenograft growth, and this effect could be decreased by irbesartan or everolimus

A. The graphs show tumor volumes with time after treatment with vehicle control, angiotensin II, angiotensin II plus irbesartan, and angiotensin II plus everolimus, respectively. *Significant difference between angiotensin II group and vehicle control group (P<0.05). **Significant difference between angiotensin II group and angiotensin II plus irbesartan, angiotensin II plus everolimus, and vehicle control groups (P<0.05). B. The immunohistochemistry of AT1R and p-mTOR in xenograft tumors in vehicle control, angiotensin II, angiotensin II plus irbesartan, and angiotensin II plus everolimus groups. The magnified figures are shown in the upper right corner. Original magnification ×200.

Figure 6. Inhibitory effect of irbesartan in 4-NQO-induced ESCC murine model

A. The incidence of esophageal tumor in mice treated with irbesartan was significantly lower than that in mice treated with vehicle control (57% versus 89%; P = 0.034). B. Gross appearance of esophagus from representative mice treated with irbesartan or vehicle control. The arrows indicate an enlargement of esophageal tumor. C. Hematoxylin and eosin stained (H&E) sections from representative mice treated with vehicle control showed esophageal squamous cell carcinoma with muscle invasion. H&E sections from representative mice treated with irbesartan showed only esophageal dysplasia. Compared to the vehicle control group, immunohistochemistry revealed lower AT1R and p-mTOR expression in the irbesartan group. The magnified figures are shown in the upper right corner. Original magnification ×200. SCC: squamous cell carcinoma.