The Hypofunctional Effect of P335L Single Nucleotide Polymorphism on SSTR5 Function

Abstract

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...

Metformin Restrains Pancreatic Duodenal Homeobox-1 (PDX-1) Function by Inhibiting ERK Signaling in Pancreatic Ductal Adenocarcinoma.

“…The evidence includes: 1) metformin inhibited PDX-1 transactivity; 2) metformin reduced PDX-1 protein and mRNA levels; and 3) metformin abrogated the increase in PDX-1 expression induced by EGF, a potent growth factor for PDAC cells. In pancreatic β cells, PDX-1 expression is subject to positive regulation by glucose, glucagon-like pepetide (GLP-1) [36, 37] and palmitic acid [38] as well as negative regulation by DNA damage [39], oxidative stress [40], somatostatin receptor 5 (SSTR5) agonist PRL-1980 [41] and advanced glycation end-products (AGEs) [42] via different mechanisms. It is not known whether these signals also operate in PDAC cells where PDX-1 acts as a pro-oncogenic transcription factor.…”

SSTR5 P335L monoclonal antibody differentiates pancreatic neuroendocrine neuroplasms with different SSTR5 genotypes

“…Among them, SSTR5 P335L SNP results from a C to T change at the 1004 th nucleotide of the human SSTR5 gene. It has been shown that SSTR5 P335L SNP is associated with neuropsychiatric diseases [11,12], pituitary adenomas [13] and pancreatic cancer [14,15]. Our recent studies also show that SSTR5 P335L is a hypofunctional SNP and, thus, could have a harmful effect on the normal functions of SSTR5 [15].…”

“…The hypofunctional property of SSTR5 P335L SNP [15] and its association with human diseases [11–14] strongly suggest that SSTR5 P335L is a potential biomarker for these human diseases. Therefore, we generated an anti-SSTR5 P335L mAb that shows specificity to SSTR5 P335L, but not WT SSTR5, overexpressed in HEK293 cells [15].…”

“…Both CPAN-1 and PANC-1 cells were grown and maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin. Expression of SSTR5 and SSTR5 P335L in CAPAN-1 and PANC-1 cells was determined by western blotting against a polyclonal anti-SSTR5 [16] (1:500) and a monoclonal anti-SSTR5 P335L [15] (2 μg/ml) antibody, respectively, using enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences Corp, Piscataway, NJ) according to the manufacturer’s protocols.…”

Scaffold Architecture Controls Insulinoma Clustering, Viability, and Insulin Production

“…Additionally, such diagnostics allow for the down-selection of the most efficacious, safest treatments prior to testing on animal models. Unfortunately, most of these in vitro evaluations are performed using cells seeded onto standard tissue culture plastic (TCP) [1][2][3][4] or biologically functionalized or coated TCP. 5,6 Though 2D studies can provide useful information, these substrates lack physical homology with the native extracellular matrix, which can alter cell behavior and reduce the ability to translate laboratory findings to the clinic.…”