49: Discovery of SMAD4 Promoter 16 Kb Upstream From its Translation Start Site

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS sions: Our findings indicate that Src kinase inhibition has antitumor effects in pancreas cancer by decreasing tumor cell motility and invasion. Inhibition of the signaling pathways involved in this process appears to be controlled by restoring ␤-catenin and E-cadherin. This suggests that Src kinase may be a promising therapeutic target in pancreatic cancer. 48. BLOCKADE OF TUMOR SUPPRESSOR PHLPP BY EGF. Y. Wang, T. Shen, X. Feng, X. Lin; Baylor College of Medicine, Houston, TX The PI3K/Akt signaling pathway controls a plethora of key cellular functions, including cell proliferation and differentiation. The central player Akt is activated by lipid PIP3, which generated by PI3K and removed by tumor suppressor PTEN. Excessive PI3K/Akt activity or loss of PTEN is often associated with tumorigenesis. Full activation of Akt also requires its phosphorylation at T308 and S473 by PKD and mTOR, respectively. Recently, a novel protein phosphatase PHLPP is identified as a negative regulator of Akt by dephosphorylating S473. Since the PI3K/Akt signaling is tightly regulated, we hypothesize that PHLPP is also under stringent control to allow cell proliferation. Hence, it is vitally important to identify how PHLPP responds to growth factor signals to regulate Akt signaling. Objective: (1) To determine whether PHLPP is phosphorylated in response to mitogenic growth factor such as EGF, and (2) to investigate how the phosphorylation regulates the functions of PHLPP. Methods: PHLPP was first identified as a phospho-protein by immunoprecipitation (IP) using anti-PHLPP antibody and then Western blotting using phospho-tyrosine antibody PY20. The specific phosphorylated tyrosine residue was determined by using PCRbased mutagenesis in the PHLPP cDNA. Expression plasmids for wild-type and mutant PHLPP were transfected in cells and their phosphorylation status was analyzed upon EGF treatment. PHLPP, the phospho-tyrosine mutant, and a series of deletion mutants of PHLPP were examined for their ability to dephosphorylate Akt, and to affect cell proliferation. Cell proliferation was measured by MTT assays and 3H-thymidine incorporation. Results: We discovered that EGF could stimulate tyrosine phosphorylation of PHLPP at position Tyr-1712. Point mutation of Tyr to Phe at amino acid 1712 (Y1712F) abolishes the phosphorylation of PHLPP. A phosphospecific antibody was then developed that could recognize the phospho-Y1712 and facilitate the detection of phospho-PHLPP. We found indeed anti-phospho-PHLPP could detect phosphorylation of wildtype PHLPP, but not Y1712F, in the presence of EGF. Importantly, the ability of Y1712F to dephosphorylate Akt was altered, suggesting that this phosphorylation site regulates the activity of PHLPP towards Akt dephosphorylation. Further experiments are under way to elucidate the molecular mechanism for how Y1712 phosphorylation controls PHLPP activity. Conclusion: PHLPP is a novel regulator of the critical PI3K/Akt signaling pathway. Our study reveals that PHLPP itself is also under tight control through tyrosine phosphorylation, and implicates that controlling PHLPP activity may represent a new way to prevent and treat human cancer.

ONCOLOGY 2: GENOMICS 49. DISCOVERY OF SMAD4 PROMOTER 16 KB UPSTREAM FROM ITS TRANSLATION START SITE. D. Calva, S. Chinnathambi, J. R. Howe, V; University of Iowa, Iowa City, IA Background: SMAD4 is the common intracellular mediator of the TGF-␤ superfamily pathway. Inactivation of SMAD4 has been linked to pancreatic adenocarcinoma, non-small cell lung cancer, colorectal cancer, and germline mutations cause the autosomal dominant cancer syndrome Juvenile Polyposis (JP). The location of the SMAD4 promoter has not been clearly defined, but knowledge of its genomic structure suggests it most likely lies upstream of 2 non-coding exons

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rather than the first coding exon, as previously thought. Since little is known about the transcription regulatory mechanisms of this important tumor suppressor gene, we set out to identify and characterize the promoter of SMAD4. Methods: Genomatix software was used to analyze the genomic region 5’ to the SMAD4 translation start site, to select the sequence most likely to have promoter activity. Deletion constructs were cloned into firefly luciferase vectors, then co-transfected into embryonic kidney and colon fibroblast cell lines with a Renilla control vector. Promoter activity was evaluated using luciferase reporter assays. MatInspector software was then used to identify regulatory binding sites. DNA sequencing of this region was performed in select JP probands by a PCR-based dideoxy chain termination method. Results: Figure 1 summarizes the luciferase activity of these constructs. A ⬎45% reduction in activity was seen between the 389 bp and 655 bp constructs; this region contains a TATA box and an SP1 site. There was a ⬎85% drop between the 189 bp and 655 bp constructs, for an overall 7-fold reduction in luciferase activity in the colon fibroblast cell line and 20-fold reduction in the embryonic kidney cell line. Two AP-2 sites were identified between both the 389-309 bp and 309-189 bp sequences. This region is located immediately upstream of the first non-coding exon of SMAD4, which lies 16.8 Kb 5’ to the translation start site in the first coding exon. Sequencing of this entire region in 35 JP probands without alterations in the coding exons of either SMAD4 or BMPR1A revealed no germline mutations. Conclusions: Strong evidence is provided by computational methods and expression vectors that a 655 bp region upstream from the first non-coding exon is the promoter of SMAD4. Although no mutations were found within the promoter region in JP patients, alteration of transcription factors or epigenetic modifications of this region could prove to play an important role in JP or sporadic cancer development.

50. THE IDENTIFICATION OF NUMEROUS SOMATIC AND GERMLINE MUTATIONS OF PDX-1 IN PANCREATIC CANCER USING GENOMIC ANALYSIS. M. C. Gingras, S. H. Liu, G. Zhou, M. Li, R. Catania, W. E. Fisher, R. A. Gibbs, F. C. Brunicardi; Baylor College of Medicine, Houston, TX Introduction: Pancreatic-duodenal homeobox 1 (PDX-1) is a transcription factor vital in pancreas development and in the regulation and expression of insulin, glucose transporter-2, glucokinase, and somatostatin. We recently demonstrated that PDX-1 regulates proliferation and invasion of pancreatic cancer cells and that it is overexpressed in pancreatic cancer. The purpose of this study was to analyze the genomic profile of PDX-1 to determine whether germline or somatic mutations are present in patients with pancreatic cancer. Methods: 45 patients diagnosed with exocrine and ampullary pancreatic adenocarcinoma were consented following an IRB approved protocol. During their surgical resection, patient blood was directly collected in PAXgene Blood DNA tubes and the DNA was isolated using the PAXgene Blood DNA kit (PreAnalytiX). Tumor specimens were processed and DNA was isolated from matched tumors using the QIAamp DNA Mini kit (Qiagen). Six primer sets were designed to cover the 2 PDX-1 exons with their surrounding intronic regions.