Introduction
Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin on insulin expression/secretion and cell proliferation. A number of single nucleotide polymorphisms (SNPs) of SSTR5 have been identified, including P335L, a non-synonymous SNP located in the protein C-terminal region and encrypted by the codons CCG (proline) or CTG (leucine). In the present study, we sought to determine if the P335L SNP affected the cellular function of SSTR5 in human pancreatic cancer as has been reported previously for neuropsychiatric diseases and pituitary adenomas.
Methods
The P335L germline genotype of 246 patients with pancreatic cancer (213 Caucasians, 16 Hispanics and 17 African Americans), and 17 human pancreatic cell lines was determined with the TaqMan SNP Genotyping assay. Human SSTR5 leucine variant (L335) was generated by performing site-directed mutagenesis using SSTR5 proline variant (P335) as a template. Transient transfections were performed in HEK293, Mia PaCa-2 and β-TC-6 cells using Lipofectamine 2000. The expression of SSTR5 L335 was determined with a mouse monoclonal anti-SSTR5 L335 antibody generated in our laboratory. The cell proliferation rate was measured by performing MTS assays. Insulin concentration was measured by performing ELISA assays.
Results
1. Genotyping of the patients' blood indicated that the frequency of the T allele (CT and TT genotypes) in codon 335 of SSTR5 in Caucasians, Hispanics and African Americans was 52%, 69% and 35%, respectively. Statistical analysis indicated no significant association existed between the frequency of the T allele and the existence of pancreatic cancer in each race. 2. Of the 17 tested human pancreatic cancer cell lines, 5 cell lines (CAPAN-2, HPAF-II, Panc03.27, Panc-1, and -3) had the homozygote TT genotype and 9 cell lines including Mia PaCa-2 were heterozygote (CT genotype). 3. Over-expression of SSTR5 L335 in Mia PaCa-2 cells enhanced cell proliferation compared to over-expression of SSTR5 P335; 4. Over-expression of SSTR5 P335 enhanced the inhibitory effect of SSTR5 agonist RPL-1980 on cell proliferation of Mia PaCa-2 cells and glucose-stimulated insulin secretion from mouse insulinoma cells, while over-expression of SSTR5 L335 blocked the inhibitory effect of RPL-1980. 5. Over-expression of SSTR5 L335 enhanced PDX-1 expression in Mia PaCa-2 cells.
Conclusion
SSTR5 P335L SNP widely exists in the human population; in patients with pancreatic cancer, which are race-dependent; and in human pancreatic cancer cell lines. In contrast to SSTR5 P335, over-expression of SSTR5 L335 variant resulted in cellular proliferation and PDX-1 over-expression in human pancreas cancer cells and blocked the inhibitory effect of an SSTR5-specific analogue on human pancreas cancer cell proliferation and glucose-stimulated insulin secretion from mouse insulinoma cells. These data suggest that SSTR5 P335L is a hypofunctional protein with a potential harmful effect on function, as well as potential latent effect, and therefore could affect...