Gastrin-releasing peptide-induced down-regulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) in neuroblastomas

Abstract

Objectives: To evaluate whether aggressive, undifferentiated neuroblastomas express tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) and to examine the effects of gastrin-releasing peptide (GRP) on PTEN gene and protein expression.

Summary background data: We have previously shown that neuroblastomas secrete GRP, which binds to its cell surface receptor (GRP-R) to stimulate cell growth in an autocrine fashion. However, the effects of GRP on expression of the tumor suppressor gene PTEN have not been elucidated in neuroblastomas.

Methods: Paraffin-embedded sections from human neuroblastomas were analyzed for PTEN and phospho-Akt protein expression by immunohistochemistry. Human neuroblastoma cell lines (SK-N-SH and SH-SY5Y) were stably transfected with the plasmid pEGFP-GRP-R to establish GRP-R overexpression cell lines, and the effects of GRP on PTEN gene and protein expression were determined.

Results: A decrease in the ratio of PTEN to phospho-Akt protein expression was identified in poorly differentiated neuroblastomas. An increase in GRP binding capacity was confirmed in GRP-R overexpressing cells, which demonstrated an accelerated constitutive cell growth rate. PTEN gene and protein expression was significantly decreased in GRP-R overexpressing cells when compared with controls.

Conclusions: Our findings demonstrate decreased expression of the tumor suppressor protein PTEN in more aggressive undifferentiated neuroblastomas. An increase in GRP binding capacity, as a result of GRP-R overexpression, down-regulates PTEN expression. These findings suggest that an inhibition of the tumor suppressor gene PTEN may be an important regulatory mechanism involved in GRP-induced cell proliferation in neuroblastomas.

FIGURE 1. PTEN/pAkt protein expression in neuroblastomas. A, A representative histologic section from differentiated (n = 10) versus undifferentiated (n = 14) neuroblastomas demonstrating differential PTEN protein expression by immunohistochemical staining (×100). An increased expression of PTEN protein (shown by the brown staining) is noted in differentiated neuroblastomas when compared with undifferentiated tumors. In contrast, phosphorylated Akt (pAkt) protein expression was not significantly altered. B, Scoring index for PTEN protein expression [scale of 1 (low) to 5 (high) was used by a blinded pathologist] (mean ± SEM; * P < 0.05 versus differentiated tumors).

FIGURE 2. GRP-R-overexpressing SK-N-SH and SH-SY5Y neuroblastoma cells. A, A representative image demonstrating increased cell cytoplasmic membrane green fluorescence with GRP-R overexpression (GRP-R) when compared with control cells (GFP alone) in both SK-N-SH and SH-SY5Y cells. B, Competition binding assay for GRP using ¹²⁵I-labeled GRP demonstrates an increased GRP-R binding capacity in both cell lines (mean ± SEM; *P < 0.05 versus GFP).

FIGURE 3. GPR-R overexpression decreases in PTEN gene expression. A, Gene array blot demonstrating significantly decreased PTEN gene expression (boxed areas) in GRP-R-overexpressing SK-N-SH cells (GRP-R) when compared with controls (GFP). B, Densitometric analysis of the PTEN gene expression change relative to housekeeping gene GAPDH.

FIGURE 4. GRP-mediated increase in phospho-Akt and decrease in PTEN protein level. GRP-R-overexpressing cells (GRP-R) show an increase phospho-Akt and decrease PTEN protein levels by Western blot analysis, compared with controls (GFP). Total Akt and β-actin protein levels were relatively unchanged.

FIGURE 5. Cell proliferation and BrdU incorporation assay. A, SH-SY5Y cells stably transfected to overexpress GRP-R (GRP-R) demonstrate an accelerated basal cell growth rate when compared with control cells (GFP vector alone) by day 4. This stimulated growth rate was attenuated with inhibition of PI3-K (LY294002; 10mM) (GRP-R/LY294002). B, When SH-SY5Y cells were transiently transfected with PTEN plasmid, the DNA synthesis rate was significantly decreased when compared with control at 24-hour time point (mean ± SEM for triplicate determination; *P < 0.05 versus control).