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

Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA.
Surgery (Impact Factor: 3.38). 12/2011; 150(6):1136-42. DOI: 10.1016/j.surg.2011.09.044
Source: PubMed


Somatostatin receptor type 5 (SSTR5) P335L is a hypofunctional, single nucleotide polymorphism of SSTR5 with implications in the diagnostics and therapy of pancreatic neuroendocrine neoplasms. The purpose of this study is to determine whether a SSTR5 P335L-specific monoclonal antibody could sufficiently differentiate pancreatic neuroendocrine neoplasms (PNENs) with different SSTR5 genotypes.
Cellular proliferation rate, SSTR5 mRNA level, and SSTR5 protein level were measured by performing MTS assay, a quantitative reverse transcription polymerase chain reaction study, Western blot analysis, and immunohistochemistry, respectively. SSTR5 genotype was determined with the TaqMan SNP Genotyping assay (Applied Biosystems, Foster City, CA).
We found that the SSTR5 analogue RPL-1980 inhibited cellular proliferation of CAPAN-1 cells more than that of PANC-1 cells. Only PANC-1 (TT) cells, but not CAPAN-1 (CC) cells expressed SSTR5 P335L. In 29 white patients with PNENs, 38% had a TT genotype for SSTR5 P335L, 24% had a CC genotype for WT SSTR5, and 38% hada CT genotype for both SSTR5 P335L and WT SSTR5. Immunohistochemistry using SSTR5 P335L monoclonal antibody detected immunostaining signals only from the neuroendocrine specimens with TT and CT genotypes, but not those with CC genotypes.
A SSTR5 P335L monoclonal antibody that specifically recognizes SSTR5 P335L but not WT SSTR5 could differentiate PNENs with different SSTR5 genotypes, thereby providing a potential tool for the clinical diagnosis of PNEN.

Download full-text


Available from: Giovanni Paganelli
  • Source
    • "The SNP widely exists in the human population and in patients with pancreatic cancer (Li et al., 2011; Zhou et al., 2011c) and pancreatic neuroendocrine tumors (Zhou et al., 2011b), which are race-dependent. SSTR5 P335L acts as a hypofunctional SNP since SSTR5 P335L enhances cell proliferation in contrast to wild-type SSTR5 (Zhou et al., 2011c). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Somatostatin (SST) is a regulatory peptide and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells. SST's actions are mediated by a family of seven transmembrane domain G protein-coupled receptors that comprise five distinct subtypes (SSTR1-5). SSTR5 is one of the major SSTRs in the islets of Langerhans. Homeodomain-containing transcription factor pancreatic and duodenal homeobox-1 (PDX-1) is essential for pancreatic development, β cell differentiation, maintenance of normal β cell functions in adults and tumorigenesis. Recent studies show that SSTR5 acts as a negative regulator for PDX-1 expression and that SSTR5 mediates somatostatin's inhibitory effect on cell proliferation and insulin expression/excretion through down-regulating PDX-1 expression. SSTR5 exerts its inhibitory effect on PDX-1 expression at both the transcriptional level by down-regulating PDX-1 mRNA and the post-translational level by enhancing PDX-1 ubiquitination. Identification of PDX-1 as a transcriptional target for SSTR5 may help in guiding the choice of therapeutic cancer treatments.
    Full-text · Article · Jun 2014 · Frontiers in Physiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin and its analogs on insulin expression/secretion and islet cell proliferation. We provide biochemical and genetic evidence that SSTR5 exerted its physiological actions via down-regulating pancreatic and duodenal homeobox-1 (PDX-1), a β-cell-specific homeodomain-containing transcription factor. Cotransfection of SSTR5 with PDX-1 resulted in dose-dependent inhibition of PDX-1 expression in human embryonic kidney 293 cells. SSTR5 agonist RPL-1980 inhibited PDX-1 expression and abolished glucagon-like peptide 1-stimulated PDX-1 expression in mouse insulinoma β-TC-6 cells. SSTR5 knockdown by short hairpin RNA led to increased PDX-1 expression that was accompanied by enhanced insulin secretion stimulated by high glucose in β-TC6 cells and alternated expressions of cell cycle proteins that favor cell proliferation in mouse insulinoma MIN6 cells. Quantitative RT-PCR analysis showed that cotransfected SSTR5 inhibited PDX-1 mRNA expression, whereas knockdown of SSTR5 increased PDX-1 mRNA expression. In addition, we found that cotransfected wild-type SSTR5 increased PDX-1 ubiquitination in human embryonic kidney 293 cells, whereas SSTR5 P335L, a hypofunctional single nucleotide polymorphism of SSTR5, inhibited PDX-1 ubiquitination. SSTR5 knockout resulted in increased expression of PDX-1, insulin, and proliferating cell nuclear antigen in the islets of sstr(-/-) mice. Immunohistochemistry analysis showed that SSTR5 P335L was associated with elevated expression of PDX-1 in human pancreatic neuroendocrine tumor. Taken together, our studies demonstrated that SSTR5 is a negative regulator for PDX-1 expression and that SSTR5 may mediate the inhibitory effects of somatostatin and its analogs on insulin expression/secretion and cell proliferation via down-regulating PDX-1 at both transcriptional and posttranslational levels.
    No preview · Article · Jun 2012 · Molecular Endocrinology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Transcription factor pancreatic and duodenal homeobox-1 (PDX-1) plays an essential role in pancreatic development, β-cell differentiation, maintenance of normal β-cell function and tumorigenesis. PDX-1 expression is tightly controlled through a variety of mechanisms under different cellular contexts. We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect. NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA. Cre activation-induced specific expression of NICD in islet β cells of transgenic βNICD+/+ mice induced increased expression of PDX-1, insulin and proliferating cell nuclear antigen (PCNA) and decreased expression of p27 with accompanied fasting hyperinsulinemia and hypoglycemia and altered responses to intraperitoneal glucose tolerance test. Systemically delivered NICD shRNA suppressed islet expression of PDX-1 and reversed the hypoglycemia and hyperinsulinemia. Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor. Thus, Notch1 acts as a positive regulator for PDX-1 expression, cooperates with PDX-1 in the development of insulin overexpression and islet cell neoplasia and represents a potential therapeutic target for islet neoplasia.
    Full-text · Article · Sep 2013 · Genes