A Unique TPA-Induced Trans-Membrane Protein Expression in Human Pancreatic Carcinoma Cells: A Novel Gene Involved in Pancreatic Cancer Growth through P42/44 MAPK
X Ding, MR Salabat, DJ Bentrem, MJ Strouch, RH Bell, TE Adrian, G-Y Yang. Northwestern University Feinberg School of Medicine, Chicago, IL
Background: Pancreatic cancer is one of the most devastating human malignancies. Mechanisms for the uncontrolled pancreatic cancer growth are poorly understood. We have cloned a novel gene induced by tetradecanoylphorbol-acetate (TPA) in pancreatic cancer cells. The gene is located on human chromosome 3, and cloning of its full-length cDNA revealed that it encodes a putative transmembrane protein with 217 amino acids. In silico analysis suggested that the gene product is localized to the endoplasmic reticulum (ER), therefore, we named this gene product TPA-induced trans-membrane protein (TTMP). The current study was to investigate its expression profile, intracellular localization and modification, its role in pancreatic cancer growth and possible signal pathways involved.
Design: TTMP expression in human pancreatic adenocarcinoma, pancreatic cancer cell lines, immortalized pancreatic ductal cells and normal human tissue was determined by real time RT-PCR. TTMP cDNA was cloned into Lentiviral expression system. Intracellular localization and post-translation modification of TTMP was investigated by immunofluorescence microscopy and deglycoslytion. Effect of TTMP on cancer cell proliferation in vitro was determined by TTMP overexpression with lentivirus and downregulation with siRNA. SCID mice were used to evaluate effect of TTMP on tumor growth following Lentiviral transduction.
Results: TTMP is widely expressed in normal human tissues and is particularly abundant in pancreas. Decreased expression of TTMP was found in pancreatic carcinoma tissue (31 cases). The TTMP protein was localized to endoplasmic reticulum membrane and had post-translational N-glycosylation. Over-expression of TTMP suppressed pancreatic cell proliferation and induced G1 phase arrest. Shut-down of the TTMP gene product by siRNA enhanced tumor cell proliferation. By implanting TTMP lentiviral-transduced pancreatic cancer cells into SCID mice, we further confirmed the in vitro results that TTMP gene product inhibited pancreatic cancer growth. Furthermore, over-expression of TTMP significantly decreases P42/44 MAPK phosphorylation.
Conclusions: The TTMP is a glycosylated protein and localizes to ER. It is involved in regulation of pancreatic adenocarcinoma growth, possibly via the P42/44 MAPK pathway. Our results further suggest that TTMP is a novel tumor suppressor gene and plays important role in pancreatic carcinogenesis.
Tuesday, March 23, 2010 2:15 PM
Platform Session: Section H, Tuesday Afternoon