Neurotensin Receptor 3/Sortilin Contributes to Tumorigenesis of Neuroendocrine Tumors Through Augmentation of Cell Adhesion and Migration

Abstract

Neurotensin (NTS), a 13-amino acid peptide which is distributed predominantly along gastrointestinal tract, has multiple physiologic and pathologic functions, and its effects are mediated by three distinct NTS receptors (NTSRs). Overexpression and activation of NTS signaling components, especially NTS and/or NTSR1, are closely linked with cancer progression and metastasis in various types of cancers including neuroendocrine tumors (NETs). Although deregulation of NTSR3/sortilin has been implicated in a variety of human diseases, the expression and role of NTSR3/sortilin in NETs have not been elucidated. In this study, we investigated the expression and oncogenic effect of NTSR3/sortilin in NETs. Increased protein levels of NTSR3/sortilin were noted in the majority of human clinical NETs (n=21) by immunohistochemical analyses compared with normal tissues (n=12). Expression of NTS and NTSR3/sortilin was also noted in all tested NET cell lines. In addition, small interfering RNA-mediated knockdown of NTSR3/sortilin decreased cell number without alteration of cell cycle progression and apoptosis induction in NET cell lines BON and QGP-1. Moreover, silencing of NTSR3/sortilin significantly suppressed cell adhesion and cell migration with inhibition of focal adhesion kinase and Src phosphorylation in the NET cells. Our results demonstrate increased expression of NTSR3/sortilin in NET patient tissues and a critical role of NTSR3/sortilin on NET cell adhesion and migration suggesting that NTSR3/sortilin contributes to NET tumorigenesis.

Figure 1

Expression analysis of NTSR3/sortilin in clinical NET tissues and four NET cell lines. (A) Human normal (nonneoplastic, n = 12) and NET (n = 21) tissue sections were stained with anti-NTSR3/sortilin antibody. Immunoreactivity scores were determined by multiplication of the values for staining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and for percentage of positive staining cells (0, no positive; 1, 0%-10% positive; 2, 11%-50% positive; 3, 51%-100% positive); *P < .05 versus normal tissue. (B) Representative images are shown for IgG (upper) or NTSR3/sortilin (bottom) staining in normal and lung NET tissues. (C) Relative expression of mRNA levels for NTS (left) and NTSR3/sortilin (right) in NET cells was assessed by qRT-PCR (*P < .05 versus BON). (D) Analysis of protein expression for NTS, NTSR3/sortilin, and β-actin in NET cells was performed by Western blot analysis. β-Actin was used as an internal control for protein loading.

Figure 2

The effect of NTSR3/sortilin knockdown on proliferation and survival of NET cells. (A) Equal numbers of BON and QGP-1 cells transfected with siRNA against control or NTSR3/sortilin were plated in 24-well plates. Cell numbers were counted in triplicate after 48- and 96-hour incubation using a cell counter (left; *P < .05 versus control siRNA). Expression levels of PCNA, a marker for proliferation, or PARP, a marker for apoptosis, and NTSR3/sortilin were measured by Western blotting using siRNA-transfected NET cells (right). (B) Flow cytometry analysis with siRNA-transfected BON and QGP-1 cells. The percentage of cells in G1, S, and G2/M phases is shown. (C) Apoptosis assays were performed in quadruplicate using Cell Death Detection ELISA^plus (Roche, Indianapolis, IN).

Figure 3

The influence of NTSR3/sortilin knockdown on NET cell adhesion. The same number of siRNA-transfected BON (A, C) and QGP-1 (B, D) cells was added directly onto cell culture plates for 6 hours (A, B) or type I collagen–coated plates for 30 minutes (C, D). The attached cells were fixed and then stained with crystal violet. Microscopic examination of the attached BON and QGP-1 cells in 48-well plates (left). The number of attached cells was counted in triplicate, and the mean values were determined (right; *P, < .05 versus control siRNA).

Figure 4

The effect of NTSR3/sortilin silencing on migration of NET cells. Migration analyses using Boyden chambers were performed using control and NTSR3/sortilin knockdown BON cells for 24 (A) and 48 (B) hours. Phase-contrast microscopic images (left) and quantification of migrated cells observed in four different fields using an inverted microscope (right) are shown (*P < .05 versus control). Using control and NTSR3/sortilin knockdown QGP-1 cells, migration assays were also performed at 24 (C) and 48 (D) hours.

Figure 5

Regulation of cell adhesion and motility modulating proteins by NTSR3/sortilin knockdown in NET cells. BON (left) and QGP-1 (right) cells were transfected with siRNA directed to NTSR3/sortilin or control. Protein was extracted, and the cell lysates were analyzed by Western blotting using antibodies against NTSR3/sortilin, FAK, phospho-FAK (Tyr397), Src, phospho-Src (Tyr416), or β-actin.