Secretin receptor promotes the proliferation of endocrine tumor cells via the PI3K/AKT pathway

Abstract

The secretin receptor (SR), a G protein-coupled receptor, mediates the effects of the gastrointestinal hormone secretin on digestion and water homeostasis. Recently, high SR expression has been observed in pancreatic ductal adenocarcinomas, cholangiocellular carcinomas, gastrinomas, and bronchopulmonary carcinoid tumors. Receptor overexpression associates with enhanced secretin-mediated signaling, but whether this molecule plays an independent role in tumorigenesis is currently unknown. We recently discovered that pheochromocytomas developing in rats affected by the MENX (multiple endocrine neoplasia-like) syndrome express at very high-level Sctr, encoding SR. We here report that SR are also highly abundant on the membranes of rat adrenal and extraadrenal pheochromocytoma, starting from early stages of tumor development, and are functional. PC12 cells, the best characterized in vitro pheochromocytoma model, also express Sctr at high level. Thus, we used them as model to study the role of SR in neoplastic transformation. Small interfering RNA-mediated knockdown of Sctr decreases PC12 cells proliferation and increases p27 levels. The proproliferative effect of SR in PC12 cells is mediated, in part, by the phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (AKT) pathway. Transfection of Sctr in Y1 adrenocortical carcinoma cells, expressing low endogenous levels of Sctr, stimulates cell proliferation also, in part, via the PI3K/AKT signaling cascade. Because of the link between SR and PI3K/AKT signaling, tumor cells expressing high levels of the receptor (MENX-associated primary pheochromocytoma and NCI-H727 human bronchopulmonary carcinoid cells) respond well and in a SR-dependent manner to PI3K inhibitors, such as NVP-BEZ235. The association between SR levels and response to PI3K inhibition might open new avenues for the treatment of tumors overexpressing this receptor.

Fig. 1.

Assessment of SR expression in serial rat adrenal gland tissue sections by receptor autoradiography. A, Hematoxyliln and eosin-stained tissue sections showing in the upper panels, A normal rat adrenal gland from a wild-type animal, the gland of a 2-month-old mutant rat, the gland of a 7-month-old mutant rat, and a paraganglioma from an 8-month-old mutant rat. The middle panels are autoradiograms showing total binding of ¹²⁵I-[Tyr¹⁰] rat secretin to the tissues shown above. Very strong and homogeneous binding to the adrenomedullary tumor cells and paraganglioma cells in older mutant rats is observed, whereas glands of young mutant rats show heterogeneous binding. The lower panels represent autoradiograms showing nonspecific binding of ¹²⁵I-[Tyr¹⁰] rat secretin in the presence of 100 nm cold human secretin. Complete displacement of ¹²⁵I-[Tyr¹⁰] rat secretin by cold secretin provides proof of specific receptor binding in all four tissues. B and C, Representative pharmacological competition experiments showing displacement of ¹²⁵I-[Tyr¹⁰] rat secretin by cold secretin, VIP, and glucagon (1-29) in (B) a pheochromocytoma and (C) pancreatic acini (control).

Fig. 2.

Down-regulation of Sctr inhibits proliferation and induces apoptosis of PC12 cells. A, PC12 cells were transfected with scrambled (negative control) or 125 ng/250 ng of anti-Sctr siRNA oligos. Cell proliferation was assessed 24 h later by measuring ATP levels. B, In parallel to A, Caspase 3/7 activity was measured to monitor apoptosis. C, In parallel to A, RNA from the transfected PC12 cells was extracted, and TaqMan RT-PCR was performed to monitor the expression level of p27. For A and B, data were analyzed independently with six replicates each and were expressed as the mean ± sem; *, P < 0.05, vs. scrambled control.

Fig. 3.

Role of SR in neurite outgrowth in PC12 cells and activation of downstream pathways. A, PC12 cells were plated on coverslips in 24-well plates, transfected with 250 ng of anti-Sctr siRNA oligos or Myc-Sctr plasmid or left untransfected. The next day, cells were incubated for additional 24 h with 100 nm secretin, fixed, and processed for immunofluorescent staining with antineurofilament high molecular weight protein (H) antibody. B and D, PC12 cells were transfected as in A and 24 h later incubated with 100 nm secretin. At the indicated time points, proteins were extracted, and Western blotting was performed to monitor P-p24/p44 MAPK (Thr202/Tyr204)/total-p42/p44 MAPK (B) and P-AKT (Ser473)/total-AKT (D) expression. C, PC12 cells were transfected as in A, and 24 h later, we assessed p27, P-AKT (Ser473), AKT, and α-tubulin expression levels by Western blotting. B–D, The ratios of the band intensities for phosphorylated proteins vs. total proteins, or for p27 vs. α-tubulin, normalized against the ratio in the untreated, scrambled-transfected control (ratio = 1), are reported below the panels.

Fig. 4.

Overexpression of Sctr promotes cell growth and activates downstream pathways in Y1 cells. A, Semiquantitative RT-PCR was performed using primer specific to the mouse Sctr. The level of mRNA was normalized against Gadph. B, Y1 cells were incubated with 1 μg of mock vector or Myc-Sctr plasmid. Cell proliferation was assessed 24 h later by measuring ATP levels. C, In parallel to B, we monitored the P-AKT, total-AKT, P-p42/p44 MAPK(Thr202/Tyr204) and total-p42/p44 MAPK, and α-tubulin expression levels by Western blotting. D, Y1 cells were transfected as in B and 24 h later incubated with 100 nm secretin. At indicated time points, proteins were extracted, and Western blotting was performed to monitor P-p24/p44 MAPK (Thr202/Tyr204)/total-p42/p44 MAPK and P-AKT (Ser473)/total-AKT expression. C and D, The ratios of the band intensities for P-AKT vs. total-AKT, normalized against the ratio in the mock-transfected control (ratio = 1), are reported below the panels. **, P < 0.01.

Fig. 5.

Effect of high SR expression on the response of Y1 cells to PI3K inhibitors. Y1 cells were transfected with mock vector or Myc-Sctr plasmid and 24 h later treated with the NVP-BKM120 (A), NVP-BEZ235 (C), or DMSO. Cell proliferation was assessed 24 h later by measuring ATP levels. *, P < 0.05; **, P < 0.01 (Myc vs. SR). B and D, In parallel samples with A and C, we assessed the p-AKT (Ser473) and total-AKT expression levels by Western blotting. The ratios of the band intensities for P-AKT vs. total-AKT, normalized against the ratio in the untreated control (CT) (ratio = 1), are reported below the panels.

Fig. 6.

Effect of SCTR down-regulation of on the response of NCI-H727 cells to PI3K inhibitors. A, Quantitative RT-PCR (TaqMan assay) for SCTR on H69 and NCI-H727 cells was performed. B, NCI-H727 cells were transfected with 250 ng of scrambled or anti-SCTR siRNA oligos and 24 h later treated with NVP-BEZ235 or DMSO. Cell proliferation was assessed 24 h later by measuring ATP levels. C, In parallel, we monitored the p-AKT (Ser473) and total-AKT expression levels by Western blotting. The ratios of the band intensities for P-AKT vs. total-AKT, normalized against the ratio in the untreated control (CT) (ratio = 1), are reported below the panels. *, P < 0.05.