Expression In Pancreatic Tumors Research Articles

Claudin and pancreatic cancer.

Due to the lack of timely and accurate screening modalities and treatments, most pancreatic cancer (PCa) patients undergo fatal PCa progression within a short period since diagnosis. The claudin(CLDN) family is expressed specifically as tight junction structure in a variety of tumors, including PCa, and affects tumor progression by changing the cell junctions. Thus far, many of the 27 members of the claudin family, including claudin-18.2 and claudin-4, have significantly aberrantly expression in pancreatic tumors. In addition, some studies have confirmed the role of some claudin proteins in the diagnosis and treatment of pancreatic tumors. By targeting different targets of claudin protein and combining chemotherapy, further enhance tumor cell necrosis and inhibit tumor invasion and metastasis. Claudins can either promote or inhibit the development of pancreatic cancer, which indicates that the diagnosis and treatment of different kinds of claudins require to consider different biological characteristics. This literature summarizes the functional characteristics and clinical applications of various claudin proteins in Pca cells, with a focus on claudin-18.2 and claudin-4.

MGST1 is a redox-sensitive repressor of ferroptosis in pancreatic cancer cells

Ferroptosis is a type of nonapoptotic cell death driven by lipid peroxidation. Here, we show a key role of MGST1 in inhibiting ferroptosis in cell cultures and mouse xenograft models. Ferroptosis activators induce MGST1 upregulation in human pancreatic ductal adenocarcinoma (PDAC) cell lines in an NFE2L2-dependent manner. The genetic depletion of MGST1 or NFE2L2 has a similar effect in promoting ferroptosis, whereas the re-expression of MGST1 restores the resistance of NFE2L2-knockdown cells to ferroptosis. MGST1 inhibits ferroptotic cancer cell death partly by binding to ALOX5, resulting in reduced lipid peroxidation. The expression of MGST1 is positively correlated with NFE2L2 expression in pancreatic tumors, which is implicated in the poor prognosis of patients with PDAC. These findings not only provide a valuable insight into the defense mechanism against ferroptotic cell death, but also indicate that targeting the MGST1 redox-sensitive pathway may be a promising strategy for the treatment of PDAC.

Magnetic Resonance Molecular Imaging of Extradomain B Fibronectin Improves Imaging of Pancreatic Cancer Tumor Xenografts.

The survival of pancreatic cancer patients can be greatly improved if their disease is detected at an early, potentially curable stage. Magnetic resonance molecular imaging (MRMI) of oncoproteins is a promising strategy for accurate, early detection of the disease. Here, we test the hypothesis that MRMI of extradomain-B fibronectin (EDB-FN), an abundant oncoprotein in the tumor extracellular matrix, can overcome the stromal barriers of pancreatic cancer to facilitate effective molecular imaging and detection of small tumors. Specimens of normal, premalignant, and malignant human pancreatic tissues were stained with a peptide-fluorophore conjugate (ZD2-Cy5.5) to assess EDB-FN binding and expression. MRMI with ZD2-N3-Gd(HP-DO3A) (MT218) specific to EDB-FN and MRI with Gd(HP-DO3A) were performed in three murine models bearing human pancreatic cancer xenografts, including a Capan-1 flank model, a BxPC3-GFP-Luc and a PANC-1-GFP-Luc intrapancreatic xenograft model. Tumor enhancement of the contrast agents was analyzed and compared. Staining of human tissue samples with ZD2-Cy5.5 revealed high EDB-FN expression in pancreatic tumors, moderate expression in premalignant tissue, and little expression in normal tissue. MRMI with MT218 generated robust intratumoral contrast, clearly detected and delineated small tumors (smallest average size: 6.1 mm2), and out-performed conventional contrast enhanced MRI with Gd(HP-DO3A). Quantitative analysis of signal enhancement revealed that MT218 produced 2.7, 2.1, and 1.6 times greater contrast-to-noise ratio (CNR) than the clinical agent in the Capan-1 flank, BxPC3-GFP-Luc and PANC-1-GFP-Luc intrapancreatic models, respectively (p < 0.05). MRMI of the ECM oncoprotein EDB-FN with MT218 is able to generate superior contrast enhancement in small pancreatic tumors and provide accurate tumor delineation in animal models. Early, accurate detection and delineation of pancreatic cancer with high-resolution MRMI has the potential to guide timely treatment and significantly improve the long-term survival of pancreatic cancer patients.

Clinical candidate and genistein analogue AXP107-11 has chemoenhancing functions in pancreatic adenocarcinoma through G protein-coupled estrogen receptor signaling.

Despite advances in cancer therapeutics, pancreatic cancer remains difficult to treat and often develops resistance to chemotherapies. We have evaluated a bioavailable genistein analogue, AXP107‐11 which has completed phase Ib clinical trial, as an approach to sensitize tumor cells to chemotherapy. Using organotypic cultures of 14 patient‐derived xenografts (PDX) of pancreatic ductal adenocarcinoma, we found that addition of AXP107‐11 indeed sensitized 57% of cases to gemcitabine treatment. Results were validated using PDX models in vivo. Further, RNA‐Seq from responsive and unresponsive tumors proposed a 41‐gene treatment‐predictive signature. Functional and molecular assays were performed in cell lines and demonstrated that the effect was synergistic. Transcriptome analysis indicated activation of G‐protein‐coupled estrogen receptor (GPER1) as the main underlying mechanism of action, which was corroborated using GPER1‐selective agonists and antagonists. GPER1 expression in pancreatic tumors was indicative of survival, and our study proposes that activation of GPER1 may constitute a new avenue for pancreatic cancer therapeutics.

Loss of ARID1A Expression Correlates With Tumor Differentiation and Tumor Progression Stage in Pancreatic Ductal Adenocarcinoma.

Mutations in the AT-rich interactive domain 1A gene, which encodes a subunit of the Switch/Sucrose nonfermentable chromatin remodeling complex, can result in loss of protein expression and are associated with different cancers. Here, we used immunohistochemistry to investigate the significance of AT-rich interactive domain 1A loss in 73 pancreatic ductal adenocarcinoma cases with paired paracancerous normal pancreatic tissues. The relationship between levels of the AT-rich interactive domain 1A protein product, BAF250a, and clinicopathological parameters in the 73 pancreatic cancer specimens was also analyzed. We found that the expression of AT-rich interactive domain 1A in normal pancreatic tissue was higher than that in tumor tissue. Loss of AT-rich interactive domain 1A expression in pancreatic tumors was associated with tumor differentiation (P = .002) and tumor stage (P = .048). Meanwhile, BAF250a protein levels were not related to lymph node metastasis, distant metastasis, sex, or age and were not associated with survival. Transfection of the pancreatic cancer cell lines AsPC-1 and PANC-1 with small-interfering RNA specific for AT-rich interactive domain 1A resulted in elevated messenger RNA and protein expression levels of B-cell lymphoma-2 (Bcl-2), CyclinD1, and Kirsten rat sarcoma viral oncogene (KRAS). The AT-rich interactive domain 1A expression level in the cells was increased following microRNA-31 (miR-31) inhibitor transfection. Our data provide additional evidence that AT-rich interactive domain 1A might function as a tumor suppressor gene in pancreatic carcinogenesis.

Abstract A22: Preclinical evaluation of Bmi1 inhibition in pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States with a five-year survival rate of 6% and a rising mortality rate. The majority of patients present with advanced disease that is unresponsive to chemotherapy, highlighting the need for novel therapeutic strategies. Bmi1, a member of the Polycomb Group (PcG) family of transcriptional repressors, is known to be upregulated in PDA beginning in early pre-malignancy. Elevated Bmi1 expression in pancreatic tumors has been shown to correlate with poor prognosis, increased metastasis, and chemotherapy resistance in patients. Additionally, a recent study found Bmi1 to be required for pancreatic neoplasia in a murine model of PDA. Together, these data support the hypothesis that Bmi1 promotes the growth and survival of pancreatic tumors, suggesting it may be a novel therapeutic target. Bmi1 is a critical member of the PcG PRC1 complex, where it interacts with E3 ligase Ring1B in a dose-dependent manner to promote mono-ubiquitination of H2AK119, a histone modification that results in repression of gene expression. Among the best-characterized functions of Bmi1 is its negative regulation of the cdkn2a locus, which encodes two tumor suppressor proteins and cell cycle regulators, p16Ink4a and p14Arf. This is notable, as p16Ink4a is inactivated in most pancreatic tumors, often through epigenetic mechanisms. More recently, Bmi1 was found to have an additional role in G2/M checkpoint regulation following double strand breaks (DSBs). Knockdown of Bmi1 in breast and prostate cancer cell lines treated with etoposide to induce DSBs was found to induce G2/M arrest, while Bmi1 overexpression reduced G2/M arrest. This effect was mediated by reduced ATM activation. Based on these data, we decided to evaluate the effect of inhibiting Bmi1 both in cultured pancreatic tumor cells and in vivo using a genetically engineered mouse model of pancreatic ductal adenocarcinoma. PTC596 is an orally available, small-molecule inhibitor of Bmi1 developed by PTC Therapeutics that inhibits proliferation of human and murine PDA cell lines in vitro. Cell cycle analysis of PTC596-treated PDA cells supports G2/M arrest as a mechanism of inhibition. PTC596 is also well tolerated in vivo and can successfully be delivered to tumor tissues of treated mice. We are currently carrying out an intervention study in the KPC (K-rasLSL.G12D/+; p53LSL.R172H/+; Pdx1-Cre) genetically engineered mouse model of PDA to evaluate whether treatment with PTC596, alone or in combination with gemcitabine, confers a survival advantage or alters tumor growth kinetics. Preliminary results support slowed tumor growth and elevated levels of apoptosis in tumor tissues of treated mice. Ongoing work focuses on identifying the mechanism(s) of the observed effects on pancreatic tumors using tissues from mice treated with PTC596 as well as PDA cells with inducible knockdown of Bmi1. Citation Format: Jaime A. Eberle, Haoxuan Yuan, Carmine Palermo, Stephen A. Sastra, Liangxian Cao, Young-choon Moon, Melissa Dumble, Thomas Davis, Kenneth P. Olive. Preclinical evaluation of Bmi1 inhibition in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A22.

Abstract A251: MGMT inhibition decreases survivin expression in pancreatic cancer.

Abstract Survivin is an antiapoptotic gene negatively regulated by p53 tumor suppressor. Survivin is overexpressed in human cancers and one of its functions is to reduce the efficacy of chemotherapy. We report that O6-benzylguanine (BG), a potent inhibitor of MGMT (O6-methyl guanine DNA methyl transferase), a DNA repair protein, curtails the expression of survivin in pancreatic cancer cells. We also show that BG, either alone or in combination with gemcitabine, inhibits survivin and PCNA expression both at the mRNA and protein levels in Panc1 and L3.6pl cells. Further, BG, either alone or in combination with gemcitabine, significantly decreases survivin promoter activity in these cells. We also show that these combinations decrease survivin protein expression in pancreatic tumors derived from orthotopic xenografts developed in nude mice. Immunohistochemistry confirms the reduced expression of survivin and PCNA in tumor sections after BG or BG plus gemcitabine treatments. We also show the enhanced recruitment of p53 protein to the survivin promoter in BG-treated pancreatic cancer cells. Further, BG promotes p53 function leading to growth inhibition. Taken together our data suggests that BG induced partial restoration of p53 activity suppresses survivin function and enhances gemcitabine effect in pancreatic cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A251. Citation Format: George C. Bobustuc, Anand Patel, Michael Thompson, Srivenugopal S. Kalkunte, Jacob Frick, James Weese, Santhi D. Konduri. MGMT inhibition decreases survivin expression in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A251.

Abstract B114: Next generation whole genome transcriptome SOLiD sequencing analysis of pancreatic ductal adenocarcinoma in LSL-KrasG12D/+ mice

Abstract Pancreatic cancer carries the most dismal prognosis of all solid tumors. It is the fourth leading cause of cancer death in the U.S. During the last three decades, the genetic alterations underlying pancreatic cancer have been well characterized. The identification and characterization of these cancer-related genes have increased our understanding of the genetic basis of pancreatic cancer development; but, unfortunately, this knowledge has not translated into better clinical practice since survival of patients with this disease has not improved significantly over the past two decades. Hence, to identify new targets that are differentially expressed in pancreatic cancer and that can be exploited for chemoprevention, coordinated screening efforts are necessary not only at the DNA level, but also at the RNA and protein levels. In this study, we used a strategy of genome-wide screening via next-generation sequencing with oligonucleotide ligation and detection (SOLiD) to analyze the whole transcriptome expression in pancreatic tumors (PT) from LSL-KrasG12D/+ mice in comparison to normal pancreas from wild type mice. Biomarker selection and real time PCR validation of novel genes was performed and ontology of key target genes was analyzed. Mapped reads data were analyzed using the Geospiza software package. Our results show ∼44,175,598 effective exon/intron reads in cancerous tissue vs. ∼30,220,306 in noncancerous pancreatic tissue. We have observed ∼12,202 differentially expressed genes using t-test with Benjamini and Hochberg correction in pancreatic tumors compared to wild type pancreas. Among them ∼12,008 genes were found to be up-regulated in PT. The first 32 genes (Cldn18, Serpinb5, Rik (2210407C18), Rik (1110032A04), sdcbp2, Fst, Sftpd, S100A8, Aspa, Cfi, Adam28, Rbp1, Cdh17, Dkk3, Krt20, Mcpt1, Ltf, Col8a1, Plac8, Il18r1, Mcpt2, Prap1, Exp1, P2ry10, AA467197, 110785, Flrt3, Gabrp, Nt5e, Gcnt3, Anxa8, Cyp2c65), were found to be altered by more than 100-fold, variations that might have potential roles during tumor development. Out of these 32 genes, Rik, sdcbp2, S100A8, Rbp1, Krt20, Ltf, Plac8, Il18r1, Mcpt2, Prap1, Exp1, AA467197, Anxa8, and Cyp2c65 were identified to have a lower splice index value, indicating more splicing of these mRNAs and differential splicing between tumor and normal. Most of the novel and previously reported genes having the highest Z scores (e.g., BMP-1, Notch-2, Adam 9, 10, 12, 17; Akap 7, 8, 12, 13; Akirin 2, 5-LOX, Axin 2, Cyclin A2, B1, D2, E2, F, H, K, L2, Y; FGF1, FGFr1, FGFr2; jak 1, 2, 3; Interleukin 6ra, 7, 15, 16, 18; chemokine receptor 1, 5, 8; Jak 1, 2 and 3) are known to be involved in cellular and metabolic processes. Among down-regulated genes, those associated with movement and establishment of cell/cellular entity (e.g., aquaporin 12, EGF, INS2, syncollin 2) were the most altered in pancreatic tumors according to Z-score, followed by genes affecting metabolic and cellular processes. The functional significance of the novel genes identified in this study for development of PT is being more fully explored. In summary, the whole genome transcriptome analysis has led to discovery of many altered key mRNAs in PT. These newly identified molecular changes may be useful as potential targets for PT detection and possibly for development of chemopreventive regimens. {Supported by NCI-CN-N01- 53300}. Citation Format: Naveena B. Janakiram, Altaf Mohammed, Misty F. Brewer, Allison Gillaspy, Stan Lightfoot, Vernon E. Steele, Chinthalapally V. Rao. Next-generation whole-genome transcriptome SOLiD sequencing analysis of pancreatic ductal adenocarcinoma in LSL-KrasG12D/+ mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B114.

Effect of Pemetrexed on Innate Immune Killer Cells and Adaptive Immune T Cells in Subjects With Adenocarcinoma of the Pancreas

Baseline levels of innate and adaptive immune cell functions were studied in patients with pancreatic cancer. The effects of pemetrexed were measured at 7 and 14 days after initial therapy then 14 days after combination therapy with gemcitabine. Pretherapy levels of absolute numbers of natural killer (NK) cells positively correlated with survival. Cytolytic units of NK activity correlated positively with NK cell numbers. Pemetrexed decreased NK cytolytic units to significance when combined with gemcitabine. Pemetrexed increased intracellular accumulation of interferon gamma (IFNγ) in NK cells that correlated negatively with survival. Addition of gemcitabine decreased IFNγ-producing NK cells to baseline. Memory (CD45RO*) T cells enumerated at baseline correlated negatively with survival but were decreased by pemetrexed therapy. Memory T cells were increased in subjects with greater B7-H3 expression in tumor tissue, whereas OX40*-activated total T cells and helper T-cell subset were decreased. FoxP3*, CD8* T cells correlated positively with progression-free interval and survival. In conclusion, innate NK-cell immunity and FoxP3*, CD8* T cells seemed beneficial to pancreatic cancer patients. Higher levels of B7-H3 expression in pancreatic tumors were detrimental to effective immunity. Although pemetrexed therapy increased activation of a subset of NK cells to produce IFNγ, addition of gemcitabine abated those responses, decreasing IFNγ-producing NK cells, whereas NK cells producing interleukin-2 without IFNγ at this timepoint positively correlated with survival. Innate immunity and adaptive immunity thus are important in defense against pancreatic cancer. Progression-free interval and survival were longer than observed in a phase III trial where gemcitabine preceded pemetrexed suggesting that a larger trial of pemetrexed preceding gemcitabine is warranted.

MHC class II expression in pancreatic tumors: a link to intratumoral inflammation

Major histocompatibility complex class II antigens (MHC class II) are constitutively expressed by professional antigen presenting cells and present antigenic peptides to specific CD4+ T lymphocytes. MHC class II expression, however, can also be induced on epithelial cells and in a variety of solid tumors. We tested MHC class II expression on tissue samples derived from patients with pancreatic ductal adenocarcinoma (PDAC) and pancreatic endocrine tumors (PET). Immunohistochemistry revealed MHC class II expression in 86 of 112 (76.8%) PDAC samples and in 30 of 43 (70.0%) PET samples. In PDAC and PET, MHC class II expression correlated significantly with severity and activity of intratumoral inflammation, as well as with the infiltration of CD4+ T lymphocytes. High MHC class II expression significantly correlated with a better histological grade of differentiation in PDAC. In vitro MHC class II expression could be induced on PDAC tumor cell lines by interferon-γ. These cells were then able to present the staphylococci enterotoxin B superantigen to T lymphocytes, which resulted in T cell proliferation. Our findings suggest that MHC class II expression on pancreatic tumor cells is induced by the intratumoral inflammatory reaction in pancreatic tumors.

Abstract 5324: Cathepsin E expression: A new potential target for pancreatic tumor imaging

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) shows unique high expression of Cathepsin E (CTSE). Here we assessed the usefulness of detecting CTSE activity in vivo as a prognostic biomarker for PDAC using mouse xenografts and transgenic models of PDAC. Procedures: CTSE expression in human and mouse pancreases with/without cancer were assessed by immunohistochemistry. Transgenic animal models of PDAC and xenografts of primary human PDAC tumors or establish pancreatic cancer cell lines in nude mice were injected with the CTSE selective fluorescent probe via tail vein. CTSE expressions in transgenic pancreatic tumors and xenografts were imaged using IVIS-100 optical imaging system. Negative control tumors were imaged in parallel. Tumors, organs and tissues of interest were imaged ex vivo for their associated Cath E expression. Data: Results confirmed the over expression of CTSE in pancreatic cancer patients and transgenic mouse models of pancreatic cancer. Images illustrate the capability of detecting CTSE expression within pancreatic tumors in both transgenic and xenograft mice models. In vivo and ex vivo images of PDAC were possible by targeting CTSE expression. Conclusions: Results represent the usefulness of CTSE as a potential molecular target for pancreatic cancer imaging. The elevated CTSE expression in pancreatic tumors allows in vivo detection of human PDAC xenografts and ex vivo imaging of transgenic PDAC tumors in mice. The study demonstrates CTSE as a promising target for tumor imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5324. doi:10.1158/1538-7445.AM2011-5324

Abstract 36: Human ecdysoneless protein: Regulator of cholesterol homeostasis in pancreatic cancer

Abstract The human Ecdysoneless protein (hEcd) is a human orthologue of the D.melanogaster ecdysoneless protein, which is highly conserved from insects to mammals and identified as one of the factors regulating the synthesis of the steroid hormone ecdysone. Ecd is required autonomously throughout the body and its deficiency can lead to reproductive or developmental defects. hEcd, also known as human suppressor of GCR2 gene (hSGT) since it can rescue growth defects in S.cerevisiae mutants with a deletion of the growth control regulatory genes 2 (GCR2), a transcriptional activator of the glycolytic gene expression. Recent investigations revealed that hEcd interacts with murine double minute-2 and stabilizes p53 by inhibiting mdm-2 mediated degradation of p53. Another study done on MEFs generated from Ecd conditional knockout mice showed evidence that Ecd interacts with retinoblastoma protein at the pocket domain and competes with E2F for association with hypophosphorylated Rb, thereby regulating E2F target gene expression and cell cycle progression. In this study, we have analyzed the expression of Ecd in PanIN and Pancreatic adenocarcinoma tissues as well as pancreatic cancer cell lines. The studies show that Ecd is overexpressed in pancreatic cancer samples as compared to normal pancreas. Therefore based on its role as a cell cycle regulator and its increased expression in pancreatic tumors, we hypothesized that Ecd might act as a potential modulator contributing to the pathogenesis of pancreatic cancer cells. Stable knockdown clones of Ecd were established in CD/HPAF pancreatic cancer cell line using two different shRNA constructs, which led to reduced proliferation rate in vitro and decreased tumor weight in vivo. Further, microarray analysis of genes in Ecd knocked-down vs scrambled cells followed by bioinformatics analysis revealed that the major pathway affected due to Ecd downregulation is the cholesterol biosynthetic and regulatory pathway. The genes which were affected by more than two-fold in Ecd knocked-down cells, encoded key cholesterol biosynthetic pathway enzymes such as FDFT1, NSDHL, DHCR7 and CYP51A1 along with INSIG1, a protein which acts as a cholesterol level sensor in cells. This was further confirmed by Quantitative Real-Time PCR analysis. Flow-cytometric analysis for cholesterol-specific filipin staining showed increased cholesterol content in Ecd-kd cells vs the scrambled cells. Additionally, insulin stimulated GLUT-4 membrane translocation, one of the cholesterol-dependent pathways was also found to be affected with Ecd knockdown cells showing higher glucose uptake than the scrambled cells. Cholesterol being a major component of cells and a mediator of endocytic and cell signaling pathways, we conclude that Ecd contributes to pancreatic cancer pathogenesis by disrupting multiple cholesterol-dependent pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 36.

Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Pancreatic cancer is the fourth leading cause of cancer deaths in the United States, with an overall 5-year survival rate of <5%. Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is highly resistant to conventional chemotherapies, underscoring the critical need for new molecular targets for pancreatic cancer chemotherapy. The KRAS proto-oncogene is mutated in >90% of PDAC. Protein kinase Ciota (PKCiota) is required for the oncogenic Ras-mediated transformed growth of lung cancer and intestinal epithelial cells. However, little is known about the role of PKCiota in pancreatic cancer. In this study, we evaluated the expression of PKCiota in human pancreatic cancer and the requirement for PKCiota for the transformed growth and tumorigenicity of PDAC cells. We find that PKCiota is significantly overexpressed in human pancreatic cancer, and high PKCiota expression correlates with poor patient survival. Inhibition of PKCiota expression blocks PDAC cell transformed growth in vitro and tumorigenicity in vivo. Inhibition of PKCiota expression in pancreatic tumors also significantly reduces tumor angiogenesis and metastasis. Analysis of downstream PKCiota effectors implicates the Rac1-MEK/ERK1/2 signaling axis in PKCiota-mediated transformed growth and cellular invasion. Taken together, our data show a required role for PKCiota in the transformed growth of pancreatic cancer cells and reveal a novel role for PKCiota in pancreatic cancer cell metastasis and angiogenesis in vivo. Our results strongly indicate that PKCiota will be an effective target for pancreatic cancer therapy.

Abstract B170: Downregulation of Yes-associated protein 1 (YAP1) expression reduces cell proliferation and clonogenicity of pancreatic cancer cells

Abstract Recent genetic studies in Drosophila have shown the Hippo pathway to be a potential tumor suppressor pathway that could play vital role(s) in mammalian cancer development. The Hippo pathway regulates organ size by inhibiting cell proliferation and promotes cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector that promotes cell growth as a transcription co-activator. In human cancer, YAP1 has been shown to be a candidate oncogene as the chromosomal region containing YAP1 (11q22) has been reported as amplified and over-expressed in several tumor types, such as colon, liver, and lung (Overholtzer et al. 2006 PNAS, 103: 12405–12410). To determine the role of YAP1 in the tumorigenesis and progression of pancreatic cancer, here we examine YAP1 protein expression in pancreatic tumors and investigate the phenotypic effects of YAP1 down-regulation in cultured pancreatic cancer cells. Initially, we evaluated YAP1 expression in pancreatic tumors through immunohistochemistry (IHC) using tissue microarrays (TMA) to determine relative protein abundance and localization at the tissue and cellular level. YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the cells, whereas the companion normal pancreas tissue showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell densities. YAP1 total protein expression and Ser127 phosphorylation increased concurrently with cell density in whole cell lysates and cytosolic fractions, but does not in nuclear fractions. Additionally, transfection of YAP1-targeting siRNA oligonucleotides within pancreatic cancer cell lines, BxPC-3 and PANC-1, significantly decreased cell viability and reduced cell proliferation in vitro. An additional observation was the diminished anchorage-independent growth on soft agar of BxPC-3 and PANC-1 cells upon transfection with YAP1 siRNA oligonucleotides. The number of colonies formed were approximately 5% in BxPC-3 (p&amp;lt;0.007) and 20% (p&amp;lt;0.04) in PANC-1 compared to the negative siRNA control. Furthermore, the reduction of YAP1 protein expression by siRNA oligonucleotides correlates with the reduced growth on soft agar. The silencing of YAP1 abated clonogenicity of pancreatic cancer cells, suggesting a role of YAP1 in tumorigenesis. In summary, we demonstrate that YAP1 is overexpressed in pancreatic cancer and potentially plays an important role in the tumorigenesis and growth of pancreatic cancer. (This work was supported by a grant from the National Foundation for Cancer Research.) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B170.

Downregulation of PAR‐4, a pro‐apoptotic gene, in pancreatic tumors harboring K‐ras mutation

Oncogenic ras is known to inhibit cell death and growth inhibitory genes and activate prosurvival genes. Proapoptotic gene PAR-4, has been found to be downregulated by oncogenic ras. Since pancreatic tumors harbor a high incidence of K-ras point mutations, we hypothesized that oncogenic K-ras might influence the function and expression of PAR-4. PAR-4 expression levels were analyzed in 4 established pancreatic tumor cell lines, 10 normal pancreatic tissues, 44 frozen tumor tissues and 25 paraffin-embedded pancreatic adenocarcinoma samples by Real Time RT-PCR, Western blot analysis and immunohistochemistry. K-ras mutational status was analyzed by allele-specific oligonucleotide-hybridization. Expression levels of PAR-4 were correlated with the K-ras mutational status and clinical characteristics. Further, modulation of endogenous PAR-4 was tested by transiently expressing oncogenic ras in a wild-type K-ras pancreatic cancer cell line, BxPC-3. Three cell lines with K-ras mutations showed low levels of PAR-4 when compared to a normal pancreatic tissue. Of 44 frozen tumors, 16 showed appreciable upregulation of Par mRNA and 27 showed significant downregulation of PAR-4 mRNA when compared to normal pancreatic tissue and 1 had levels equivalent to normal pancreatic tissue. Of 25 paraffin-embedded tumors, 9 showed downregulation of PAR-4 protein and this downregulation of PAR-4 correlated significantly with K-ras mutational status (p < 0.00002). In addition, the presence of PAR-4 mRNA or protein expression in pancreatic tumors correlated with prolonged survival. Transient overexpression of oncogenic ras in wild-type K-ras BxPC-3 cells significantly downregulated the endogenous PAR-4 protein levels and conferred accelerated growth. Thus, downregulation or loss of PAR-4 expression by oncogenic ras may provide a selective survival advantage for pancreatic tumors, through inhibition of proapoptotic pathway mediated by PAR-4.

Targeted Expression of BikDD Eradicates Pancreatic Tumors in Noninvasive Imaging Models

Pancreatic cancer is an aggressive malignancy with morbidity rates almost equal to mortality rates because of the current lack of effective treatment options. Here, we describe a targeted approach to treating pancreatic cancer with effective therapeutic efficacy and safety in noninvasive imaging models. We developed a versatile expression vector "VISA" (VP16-GAL4-WPRE integrated systemic amplifier) and a CCKAR (cholecystokinin type A receptor) gene-based, pancreatic-cancer-specific promoter VISA (CCKAR-VISA) composite to target transgene expression in pancreatic tumors in vivo. Targeted expression of BikDD, a potent proapoptotic gene driven by CCKAR-VISA, exhibited significant antitumor effects on pancreatic cancer and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of pancreatic tumors with virtually no toxicity.

Estrogen Receptors in Pancreatic Tumors

Although a potential role for estrogen receptors (ER) in pancreatic tumors has been debated for many years, the importance of the receptors in these neoplasms remains unknown. Even the expression of the 2 ER isoforms, ER-alpha and ER-beta, in histological subtypes of pancreatic neoplasms is controversial. The aim of the present study was to systematically review the available literature about ER expression in pancreatic tumors and to discuss the potential importance of estrogen signaling in them. We performed a comprehensive literature search and analyzed the results regarding ER expression in pancreatic tumors, with special emphasis on the specificity of the antibodies used for immunohistochemistry. Many articles have been published investigating the expression of ERs in pancreatic tumors, but the results are inconsistent. Moreover, most studies used antibodies that detected only ER-alpha, not ER-beta. Thus, the expression pattern of ER-beta in pancreatic neoplasm remains especially unclear. The lack of detailed studies evaluating the expression of both ER-alpha and ER-beta receptors using isoform-specific antibodies likely contributes to the inconsistency of published results concerning ER expression in pancreatic tumors. Available published evidence suggests that a thorough reexamination of the potential role of ERs in pancreatic neoplasms is warranted.

Cdx2 expression in pancreatic tumors: Relationship with prognosis of invasive ductal carcinomas

We have previously reported that Caudal-related homeobox gene 2 (Cdx2) is a useful prognostic intestinal phenotypic marker for advanced gastric cancers. In this study, we examined Cdx2 expression and phenotype in pancreatic tumors. We evaluated 19 mucinous cystic tumors (MCTs), 17 intraductal papillary-mucinous tumors (IPMTs), and 41 invasive ductal carcinomas (IDCs) with regard to their gastrointestinal phenotype. The expression of Cdx2 was also assessed immunohistochemically. The lesions were phenotypically divided into 39 gastric (G type), 29 gastric and intestinal mixed (GI type), 3 intestinal (I type), and 6 null (N type) types, independent of the histopathological type. Most of the pancreatic tumors were thus judged to be the positive for gastric phenotypic members. Cdx2 nuclear staining demonstrated a close relation to the intestinal phenotypic expression in all three types (MCTs, IPMTs, and IDCs; p<0.05). In IDCs, Kaplan-Meier analysis of Cdx2 expression showed the Cdx2-positive group to have a significantly better outcome than their negative counterparts (p=0.015). In conclusion, our data suggest that Cdx2 might be necessary for intestinal phenotypic expression even in pancreatic tumor cells. In addition, Cdx2 expression in IDCs may be a novel prognostic marker for patient survival.

Differential expression of the Tie-2 receptor and its ligands in human pancreatic tumors

Derangements in the balance of the Tie-2 receptor ligands, angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), have been implicated in the growth and differentiation of several human tumors. To assess the potential role of angiogenesis factors in pancreatic tumorigenesis, we confirmed previously observed oligonucleotide-based microarray data by analyzing their expression in pancreatic tumors using semiquantitative reverse transcription polymerase chain reaction and immunohistochemistry. RNA harvested from tissue samples obtained from patients with normal pancreata, adenocarcinoma of the pancreas, and neuroendocrine tumors of the pancreas (nine each) was grouped by tissue type, pooled, and hybridized to a cDNA microarray and global gene expression patterns were determined. Semiquantitative reverse transcription polymerase chain reaction was applied to individual pool components to specifically determine expression of Tie-2, Ang-1, and Ang-2. A separate set of tumors and matched normal pancreas were then stained for expression of Ang-2 protein using immunohistochemistry. Microarray data showed that Ang-2 was upregulated in neuroendocrine tumors ( approximately 8 times more than normal) and adenocarcinoma of the pancreas ( approximately 5 times more than normal) although Ang-1 and Tie-2 were not differentially expressed. Reverse transcription polymerase chain reaction confirmed these findings showing strong Ang-2 expression in nine of nine neuroendocrine tumors, eight of nine adenocarcinoma of the pancreas, and weak expression in two of nine normal pancreata. Immunohistochemistry showed tumor-specific staining of Ang-2 in 10 of 15 matched adenocarcinoma of the pancreas (67%) and 6 of 8 neuroendocrine tumors (75%). Although Tie-2 and Ang-1 are not differentially expressed in pancreatic tumors, Ang-2 gene and gene product are overexpressed, suggesting a significant role for Ang-2 in pancreatic tumorigenesis. Differential expression of Ang-2 in human tumors may be useful as a diagnostic and therapeutic target.

Human MUC4 mucin cDNA and its variants in pancreatic carcinoma.

The human MUC4 gene is not expressed in normal pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors. To investigate the tumor-associated expression, MUC4 cDNA was cloned from a human pancreatic tumor cell line cDNA expression library using a polyclonal antibody raised against human deglycosylated mucin and RT-PCR. Pancreatic MUC4 cDNA shows differences in 12 amino acid residues in the non-tandem repeat coding region with no structural rearrangement as compared with tracheal MUC4. The full-length MUC4 cDNA includes a leader sequence, a serine and threonine rich non-tandem repeat region, a central large tandem repeat domain containing 48 bp repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, EGF-like domains, and a transmembrane domain. We also report the presence of a new EGF-like domain in MUC4 cDNA, located in the cysteine-rich region upstream from the first EGF-like domain. Four distinct splice events were identified in the region downstream of the central tandem repeat domain that generate three new MUC4 cDNA sequences (sv4, sv9, and sv10). The deduced amino acid sequences of two of these variants lack the transmembrane domain. Furthermore, two unique forms of MUC4 (MUC4/Y and MUC4/X) generated as a result of alternative splicing lack the salient feature of mucins, the tandem repeat domain. A high degree of polymorphism in the central tandem repeat region of MUC4 was observed in various pancreatic adenocarcinoma cell lines, with allele sizes ranging from 23.5 to 10.0 kb. MUC4 mRNA expression was higher in differentiated cell lines, with no detectable expression in poorly differentiated pancreatic tumor cell lines.