Dawoon E. Jung

Differentially expressed microRNAs in pancreatic cancer stem cells.

Objective: The objective of this study was to analyze and identify pancreatic cancer stem cell-specific microRNAs (miRNAs) and messenger RNAs (mRNAs) to investigate their correlations to cancer stem cell biology. Methods: We used sphere cultivation methods to enrich the stem cell population and analyzed overall miRNA and mRNA expressions using microarray analysis. Results: Differentially expressed miRNAs including miR-99a, miR-100, miR-125b, miR-192, and miR-429 were detected in pancreatic cancer stem cells. Furthermore, examining both profiles, we obtained 210 miRNAs and 258 stem cell-associated mRNAs that were differentially expressed in the pancreatic cancer stem cells. These miRNAs and mRNAs were further investigated using cross-correlation analysis, which yielded 6 groups of miRNAs and 3 groups of mRNAs. The number of miRNA clusters and mRNA clusters showed high correlation based on microarray result. Conclusions: Differentially expressed miRNAs in pancreatic cancer stem cells provide insights into possible linkages between clusters of miRNAs and clusters of stem cell-associated mRNAs in cancer stem cells and have broad implications in our understanding of cancer stem cells and cancer stem cell-targeted cancer therapy.

Pancreatic adenocarcinoma up‐regulated factor (PAUF), a novel up‐regulated secretory protein in pancreatic ductal adenocarcinoma

The identification of novel tumor‐specific proteins or antigens is of great importance for diagnostic and therapeutic applications in pancreatic cancer. Using oligonucleotide microarrays, we identified a broad spectrum of differentially expressed pancreatic cancer‐related genes. Of these, we selected an overexpressed expressed sequence taq and cloned a 721‐bp full‐length cDNA with an open reading frame of 196 amino acids. This novel gene was localized on the Homo sapiens 16p13.3 chromosomal locus, and its nucleotide sequence matched the Homo sapiens similar to common salivary protein 1 (LOC124220). We named the gene pancreatic adenocarcinoma up‐regulated factor. The pancreatic adenocarcinoma up‐regulated factor was secreted into the culture medium of pancreatic adenocarcinoma up‐regulated factor‐overexpressing Chinese hamster ovary cells, had an apparent molecular mass of ~25 kDa, and was N‐glycosylated. The induction of pancreatic adenocarcinoma up‐regulated factor in Chinese hamster ovary cells increased cell proliferation, migration, and invasion ability in vitro. Subcutaneous injection of mice with Chinese hamster ovary/pancreatic adenocarcinoma up‐regulated factor cells resulted in 3.8‐fold greater tumor sizes compared to Chinese hamster ovary/mock cells. Reverse transcription–polymerase chain reaction and western blotting with antirecombinant human pancreatic adenocarcinoma up‐regulated factor antibodies confirmed that pancreatic adenocarcinoma up‐regulated factor was highly expressed in six of eight pancreatic cancer cell lines. Immunohistochemical staining of human pancreatic cancer tissues also showed pancreatic adenocarcinoma up‐regulated factor overexpression in the cytoplasm of cancer cells. Transfection with pancreatic adenocarcinoma up‐regulated factor‐specific small‐interfering RNA reduced cancer cell migration and invasion in vitro. Treatment with antirecombinant human pancreatic adenocarcinoma up‐regulated factor in vitro and in vivo reduced proliferation, migration, invasion, and tumorigenic ability. Collectively, our results suggest that pancreatic adenocarcinoma up‐regulated factor is a novel secretory protein involved in pancreatic cancer progression and might be a potential target for the treatment of pancreatic cancer. (Cancer Sci 2009; 100: 828–836)

Aberrant Hedgehog Ligands Induce Progressive Pancreatic Fibrosis by Paracrine Activation of Myofibroblasts and Ductular Cells in Transgenic Zebrafish

Hedgehog (Hh) signaling is frequently up-regulated in fibrogenic pancreatic diseases including chronic pancreatitis and pancreatic cancer. Although recent series suggest exclusive paracrine activation of stromal cells by Hh ligands from epithelial components, debates still exist on how Hh signaling works in pathologic conditions. To explore how Hh signaling affects the pancreas, we investigated transgenic phenotypes in zebrafish that over-express either Indian Hh or Sonic Hh along with green fluorescence protein (GFP) to enable real-time observation, or GFP alone as control, at the ptf1a domain. Transgenic embryos and zebrafish were serially followed for transgenic phenotypes, and investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry. Over-expression of Ihh or Shh reveals virtually identical phenotypes. Hh induces morphologic changes in a developing pancreas without derangement in acinar differentiation. In older zebrafish, Hh induces progressive pancreatic fibrosis intermingled with proliferating ductular structures, which is accompanied by the destruction of the acinar structures. Both myofibroblasts and ductular are activated and proliferated by paracrine Hh signaling, showing restricted expression of Hh downstream components including Patched1 (Ptc1), Smoothened (Smo), and Gli1/2 in those Hh-responsive cells. Hh ligands induce matrix metalloproteinases (MMPs), especially MMP9 in all Hh-responsive cells, and transform growth factor-ß1 (TGFß1) only in ductular cells. Aberrant Hh over-expression, however, does not induce pancreatic tumors. On treatment with inhibitors, embryonic phenotypes are reversed by either cyclopamine or Hedgehog Primary Inhibitor-4 (HPI-4). Pancreatic fibrosis is only prevented by HPI-4. Our study provides strong evidence of Hh signaling which induces pancreatic fibrosis through paracrine activation of Hh-responsive cells in vivo. Induction of MMPs and TGFß1 by Hh signaling expands on the current understanding of how Hh signaling affects fibrosis and tumorigenesis. These transgenic models will be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases which are induced by Hh signaling activation.

The DNA aptamer binds stemness‐enriched cancer cells in pancreatic cancer

Pancreatic cancer remains one of the most common and lethal cancers. Most patients (80%) present with inoperable advanced pancreatic cancer at initial diagnosis, and their early diagnosis is a significant unmet challenge. Recent studies indicate that cancer, including pancreatic cancer, is initiated and propagated by cancer stem cells (CSCs). CSCs are responsible not only for the pathogenesis of cancer but also for the heterogeneity, malignant degree, anticancer therapy resistance, and recurrence of tumors. Therefore, the identification of CSCs may be a crucial stepping stone for overcoming this disastrous pancreatic cancer. Here, we investigated pancreatic CSC‐associated aptamers as a novel tool for diagnosis and therapeutic agents. Aptamers that bind to stemness‐enriched cancer cells in pancreatic cancer were developed by modified Cell‐SELEX method. Positive selection was performed by the sphere cells generated by pancreatic cancer cell line, HPAC, and then the aptamer pool was negatively selected by pancreatic normal cell line, HPDE. Aptamers 1 and 146 showing high specificity upon the KD values with 22.18 and 22.62 nM were selected. These 2 aptamers were validated by binding to HPAC sphere cells and to HPDE cells, and both aptamers showed specificity to HPAC sphere cells only. Aptamer‐positive cells showed high expression levels of CSC‐associated genes compared with the aptamer‐negative cells by FACS analysis. The colocalization of CD44, CD24, ESA, and CD133 was also observed in the aptamer‐positive cells by confocal microscopy. In the present study, these 2 pancreatic CSC‐associated aptamers may be potential candidates for novel diagnostic markers, CSC‐targeting drug delivery, or circulating tumor cell detection.

Impaired Lymphocytes Development and Xenotransplantation of Gastrointestinal Tumor Cells in Prkdc-Null SCID Zebrafish Model.

Severe combined immunodeficiency (SCID) mice have widely been used as hosts for human tumor cell xenograft study. This animal model, however, is labor intensive. As zebrafish is largely emerging as a promising model system for studying human diseases including cancer, developing efficient immunocompromised strains for tumor xenograft study are also demanded in zebrafish. Here, we have created the Prkdc-null SCID zebrafish model which provides the stable immune-deficient background required for xenotransplantation of tumor cell. In this study, the two transcription activator-like effector nucleases that specifically target the exon3 of the zebrafish Prkdc gene were used to induce a frame shift mutation, causing a complete knockout of the gene function. The SCID zebrafish showed susceptibility to spontaneous infection, a well-known phenotype found in the SCID mutation. Further characterization revealed that the SCID zebrafish contained no functional T and B lymphocytes which reflected the phenotypes identified in the mice SCID model. Intraperitoneal injection of human cancer cells into the adult SCID zebrafish clearly showed tumor cell growth forming into a solid mass. Our present data show the suitability of using the SCID zebrafish strain for xenotransplantation experiments, and in vivo monitoring of the tumor cell growth in the zebrafish demonstrates use of the animal model as a new platform of tumor xenograft study.

Embigin is overexpressed in pancreatic ductal adenocarcinoma and regulates cell motility through epithelial to mesenchymal transition via the TGF-β pathway

Embigin is a member of the immunoglobulin superfamily and encodes a transmembrane glycoprotein. There have been reports of Embigin involvement in neuromuscular junction formation and plasticity; however, the molecular functions of Embigin in other organs are unknown. Our aim was to investigate the possible role of Embigin in pancreatic cancer. In pancreatic ductal adenocarcinoma tissues, Embigin expression was higher than that in normal pancreatic tissues. Immunohistochemical analysis revealed expression of Embigin in pancreatic cancer cells, as well as expression of monocarboxylate transporter 2 (MCT2) in cancer tissues. To gain further insight, we transfected BxPC‐3 and HPAC pancreatic cancer cells with siRNA or shRNA targeting Embigin and observed reductions in cell proliferation, migration, invasion, wound healing, and reduced levels of matrix metalloproteinases‐2 and ‐9. Silencing of Embigin increased intracellular L‐lactate concentration by 1.5‐fold and decreased MCT2 levels at the plasma membrane. Furthermore, Embigin silencing led to a reduced expression of PI3K, GSK3‐β, and Snail/Slug. Upon treating BxPC‐3 cells with transforming growth factor‐β (TGF‐β), we observed elevated expression of Snail/Slug, Embigin, and Vimentin; meanwhile, when treating cells with SB‐216763, a GSK3‐β inhibitor, we noted decreases in GSK3‐β, Snail/Slug, and Embigin expression, suggesting that the TGF‐β signaling cascade, comprising PI3K, GSK3‐β, Snail/Slug, and Embigin signals, mediates epithelial to mesenchymal transition (EMT) in pancreatic cancer cells. These findings indicate the involvement of Embigin in EMT in pancreatic cancer progression and suggest Embigin as a putative target for the detection and/or treatment of pancreatic cancer.

CG200745, an HDAC inhibitor, induces anti-tumour effects in cholangiocarcinoma cell lines via miRNAs targeting the Hippo pathway

Cholangiocarcinoma is a devastating malignancy with fatal complications that exhibits low response and resistance to chemotherapy. Here, we evaluated the anticancer effects of CG200745, a novel histone deacetylase inhibitor, either alone or in combination with standard chemotherapy drugs in cholangiocarcinoma cells. CG200745 dose-dependently reduced the viability of cholangiocarcinoma cells in vitro and decreased tumour volume and weight in a xenograft model. Administering CG200745 along with other chemotherapeutic agents including gemcitabine, 5-fluorouracil (5-FU), cisplatin, oxaliplatin, or gemcitabine plus cisplatin further decreased cholangiocarcinoma cell viability, with a combination index < 1 that indicated synergistic action. CG200745 also enhanced the sensitivity of gemcitabine-resistant cells to gemcitabine and 5-FU, thereby decreasing cell viability and inducing apoptosis. This was accompanied by downregulation of YAP, TEAD4, TGF-β2, SMAD3, NOTCH3, HES5, Axl, and Gas6 and upregulation of the miRNAs miR-22-3p, miR-22-5p, miR-194-5p, miR-194-3p, miR-194-5p, miR-210-3p, and miR-509-3p. The Ingenuity Pathway Analysis revealed that CG200745 mainly targets the Hippo signalling pathway by inducing miR-509-3p expression. Thus, CG200745 inhibits cholangiocarcinoma growth in vitro and in vivo, and acts synergistically when administered in combination with standard chemotherapeutic agents, enabling dose reduction. CG200745 is therefore expected to improve the outcome of cholangiocarcinoma patients who exhibit resistance to conventional therapies.

In VivoStudy of Natural Killer (NK) Cell Cytotoxicity Against Cholangiocarcinoma in a Nude Mouse Model

Background/Aim: Natural killer (NK) cells are one of the lymphocytes clinically used for various cancer types. Cytotoxicity of NK cells to cholangiocarcinoma (CC), however, has not yet been studied. Nor NK cell therapy against CC has been clinically applied. In this study, relevance of NK cell therapy for anti-tumor efficacy against CC was pre-clinically investigated. Materials and Methods: Human HuCCT-1 cells, an intrahepatic CC cell line, were xenografted into nude mice. The HuCCT-1 tumor-bearing nude mice then received multiple infusions of ex vivo-expanded human NK cells (SMT01) and in vivo cytotoxic activity of the NK cells against the CC cells was evaluated. Results: SMT01 infusion resulted in significant inhibition of the CC tumor growth. Body weight of the mice administrated with chemotherapy was found to be maintained at the lowest level among all treatment groups while all the SMT01 infusion groups well maintained their body weight. Conclusion: The present in vivo study demonstrates that NK cells contain cytolytic activity against cholangiocarcinoma and show beneficial effect of NK cell therapy in relevance to quality of life. Further investigation of the NK cell-based immunotherapy can be useful to determine cancer therapeutics for the specific tumor.

Combined use of CEMIP and CA 19-9 enhances diagnostic accuracy for pancreatic cancer

Carbohydrate antigen (CA) 19-9 is the only diagnostic marker used in pancreatic cancer despite its limitations. Here, we aimed to identify the diagnostic role of CEMIP (also called KIAA1199) combined with CA 19-9 in patients with pancreatic cancer. A retrospective analysis of prospectively collected patient samples was performed to determine the benefit of diagnostic markers in the diagnosis of pancreatic cancer. We investigated CEMIP and CA 19-9 levels in 324 patients with pancreatic cancer and 49 normal controls using serum enzyme-linked immunosorbent assay. Median CA 19-9 and CEMIP levels were 410.5 U/ml (40.8–3342.5) and 0.67 ng/ml (0.40–1.08), respectively, in patients with pancreatic cancer. The AUROC for CA 19-9 and CEMIP were 0.847 (95% confidence interval [CI]: 0.806–0.888) and 0.760 (95% CI: 0.689–0.831), respectively. Combination of CA 19-9 with CEMIP showed markedly improved AUROC over CA 19-9 alone in pancreatic cancer diagnosis (0.94 vs. 0.89; P < 0.0001). CEMIP showed a diagnostic yield of 86.1% (68/79) in CA 19-9 negative pancreatic cancer. Combined use with CEMIP showed significantly improved diagnostic value compared with CA 19-9 alone in pancreatic cancer. Especially, CEMIP may be a complementary marker in pancreatic cancer patients with normal CA 19-9 levels.

TSH-independent release of thyroid hormones through cold exposure in aging rats

Thyroid function decreases and cold exposure response becomes impaired with increasing age. We investigated the age-related changes in thyroid structure and function and cold-induced changes in the thyroid activity of aging rats. Thirty-two male Sprague–Dawley rats were randomly divided into four groups (8 rats per group): young (7 months) and old (22 months) groups exposed to room temperature and cold stress. The active follicle ratio and serum free T3, T4 and TSH, and TSH receptor (TSHR) concentrations in the thyroid tissues of the rats from each group were compared. At room temperature, old rats had significantly lower active follicle ratio and free T3 and T4 concentrations than young rats. Furthermore, old rats displayed higher TSH level than young. Exposure to cold temperature led to significantly increased active colloid ratio and free T3 and T4 concentrations among old rats, but no significant differences were found among young rats. Additionally, no significant changes in the TSH and TSHR levels were observed after cold exposure in both young and old rats. Old rats have lower thyroid function than young rats under normal temperature. Aging rats are more susceptible to cold stress than young rats, and cold-induced thyroid activation occurs independently of TSH. We investigated the age-related changes in the thyroid structure and function and cold-induced changes in the thyroid activity of aging rats. Aging rats have structurally less active thyroid follicles and functionally lower thyroid hormone levels than young rats. Furthermore, old rats are more susceptible to cold stress than young rats, and cold-induced thyroid activation occurs independently of TSH.