The hypofunctional effect of P335L single nucleotide polymorphism on SSTR5 function.

TitleThe hypofunctional effect of P335L single nucleotide polymorphism on SSTR5 function.
Publication TypeJournal Article
Year of Publication2011
AuthorsZhou, G, Gingras, M-C, Liu, S-H, Li, D, Li, Z, Catania, RL, Stehling, KM, Li, M, Paganelli, G, Gibbs, RA, DeMayo, FJ, Fisher, WE, F Brunicardi, C
JournalWorld J Surg
Volume35
Issue8
Pagination1715-24
Date Published2011 Aug
ISSN1432-2323
KeywordsAdenocarcinoma, African Americans, Alleles, Animals, Antibodies, Monoclonal, Cell Line, Tumor, Cell Proliferation, Codon, European Continental Ancestry Group, Gene Expression Regulation, Neoplastic, Gene Frequency, Genotype, Hispanic Americans, Humans, Insulin, Insulin Secretion, Mice, Mice, Inbred BALB C, Pancreatic Neoplasms, Polymorphism, Single Nucleotide, Receptors, Somatostatin
Abstract

BACKGROUND: 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 nonsynonymous SNP located in the protein C-terminal region and encrypted by the codon CCG (proline) or the codon CTG (leucine). In the present study we sought to determine the distribution of the SSTR5 P335L SNP in a cohort of pancreatic cancer patients and whether the P335L SNP affected cellular function of SSTR5 in human pancreatic cancer.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: 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, which was race-dependent. Statistical analysis indicated that association between the frequency of the T allele and the existence of pancreatic cancer in each race missed significance perhaps due to limited sample size. In 17 tested human pancreatic cancer cell lines, 5 (Capan-2, HPAF-II, Panc03.27, Panc-1, and -3) were homozygous (TT genotype) and 9, including Mia PaCa-2, were heterozygous (CT genotype). Overexpression of SSTR5 L335 in Mia PaCa-2 cells enhanced cell proliferation compared to overexpression of SSTR5 P335. Overexpression 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 overexpression of SSTR5 L335 blocked the inhibitory effect of RPL-1980. Overexpression of SSTR5 L335 enhanced PDX-1 expression in Mia PaCa-2 cells. A specific monoclonal antibody was generated to detect SSTR5 P335L.CONCLUSION: SSTR5 P335L SNP widely exists in the human population, in patients with pancreatic cancer, and is race-dependent. The SNP is also present in selected human pancreatic cancer cell lines. In contrast to SSTR5 P335, overexpression of the SSTR5 L335 variant resulted in cellular proliferation and PDX-1 overexpression in human pancreatic cancer cells. Its overexpression blocked the inhibitory effect of an SSTR5-specific analog on human pancreatic cancer cell proliferation and on glucose-stimulated insulin secretion from mouse insulinoma cells. These data suggest that SSTR5 P335L is a hypofunctional protein with a potentially harmful effect on function, as well as potential latent effect, and therefore it could affect the clinical response to somatostatin analog therapy for patients with pancreatic cancer.

DOI10.1007/s00268-010-0939-9
Alternate JournalWorld J Surg
PubMed ID21249361
PubMed Central IDPMC4137969
Grant ListR01 DK046441 / DK / NIDDK NIH HHS / United States
U54 HG003273 / HG / NHGRI NIH HHS / United States
R01-DK46441 / DK / NIDDK NIH HHS / United States