Primary Pancreatic Cancer Tissues Research Articles

Abstract 4006: Smad6 upregulation provides an alternative mechanism for BMP inactivation in SMAD4 wild type pancreatic cancers.

Abstract Introduction: Genetic inactivation of SMAD4, a central mediator of TGF-β superfamily signaling, is significantly correlated with metastatic behavior in pancreatic cancer patients. However, because some patients with pancreatic cancer have genetically intact TGF-β and BMP pathway components, we investigated the role of alternative mechanisms of inactivation in promoting pancreatic cancer metastasis. Methods: Eighteen cell lines for which the genetic status of all members of the TGF-β pathway were known were used in this study. TGF-β signaling levels were analyzed in each cell line using a luciferase reporter system under the control of a Smad Binding Element (SBE). Immunoblotting was performed on protein lysates from pancreatic cancer cell lines of known genotype. Cell lines that express high levels of Smad6 were transiently transfected with shRNA constructs targeting Smad6; cell lines that express low levels of Smad6 were transiently transfected with a construct to constitutively express Smad6. The effects of Smad6 modulation were assessed by proliferation assay, migration assay, and invasion assay. Immunohistochemistry was performed on primary and metastatic pancreatic cancer tissues and staining intensity correlated to clinical data. Results: Functional TGF-β and BMP signaling were eliminated in cell lines with known SMAD4 inactivation, as well as in several cell lines in which these pathways remain intact. Immunoblotting for known TGF-β superfamily antagonists in these cell lines revealed differential expression of Smad6, an inhibitory Smad. Modulation of Smad6 levels in vitro suggests that Smad6 overexpression contributes to increased levels of proliferation, migration, and invasion in SMAD4-intact cell lines. Overexpression of Smad6 did not restore TGF-β signaling, but did increase BMP response. Immunohistochemistry for Smad6 in patient samples revealed a nuclear localization pattern, suggesting that the pro-oncogenic roles of Smad6 are mediated by its ability to act as a transcription factor. High Smad6 levels correlated with worse prognosis and metastatic behavior among SMAD4-intact pancreatic cancers. Conclusions: Smad6, an inhibitory Smad, is differentially expressed in pancreatic cancer, both in cell lines and patient samples. High levels of Smad6 in patients at autopsy associate with widespread metastasis, irrespective of SMAD4 status. Preliminary studies in vitro support a metastasis-promoting function of Smad6: Smad6 overexpression is associated with increased levels of proliferation, migration, and invasion in SMAD4-intact pancreatic cancer cell lines. Experiments are ongoing to determine the mechanism through which Smad6 is acting in pancreatic cancer. Citation Format: Jacqueline A. Brosnan, Richard Morgan, Catherine M. White, Seung-Mo Hong, Shinichi Yachida, Michael Goggins, Barish Edil, Christine A. Iacobuzio-Donahue. Smad6 upregulation provides an alternative mechanism for BMP inactivation in SMAD4 wild type pancreatic cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4006. doi:10.1158/1538-7445.AM2013-4006

Clinical impact of circulating miR-221 in plasma of patients with pancreatic cancer

Background:Several recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We tested miR-221 and miR-375, which are frequently reported to be highly and poorly expressed in pancreatic cancer (PCa), as candidates for plasma biomarkers in PCa.Methods:This study was divided into three parts: (1) Confirmation of higher miR-221 levels in primary PCa tissue and cell lines than normal pancreatic tissues. (2) Evaluation of plasma miR-221 and miR-375 concentrations by comparing results from 47 consecutive PCa patients and 30 healthy volunteers. (3) Evaluation of the assay for monitoring tumour dynamics in PCa patients.Results:(1) Expression of miR-221 was significantly higher in PCa tissues and cell lines than normal pancreatic tissues. (2) Plasma miR-221 concentrations were significantly higher in PCa patients than that in benign pancreatic tumours (P=0.016) and controls (P<0.0005), while plasma miR-375 concentrations tended to be lower in PCa patients (P=0.064), and the miR-221/miR-375 ratio was significantly higher (P<0.0001) in PCa patients than in controls. (3) Plasma miR-221 concentrations were significantly reduced in postoperative samples (P=0.018). Furthermore, PCa patients with high plasma miR-221 concentrations had significant correlation with distant metastasis (P=0.041), and non-resectable status (P=0.021).Conclusion:Plasma miR-221 could be a useful biomarker for cancer detection, monitoring tumour dynamics and predicting malignant outcomes in PCa patients, and may contribute to clinical decision making in PCa treatments.

Novel Role of NOX in Supporting Aerobic Glycolysis in Cancer Cells with Mitochondrial Dysfunction and as a Potential Target for Cancer Therapy

Author SummaryGlycolysis is a cytoplasmic metabolic process that produces energy from glucose. In normal cells, the rate of glycolysis is low, and glycolysis products are further processed in the mitochondria via oxidative phosphorylation, a very efficient energy-producing process. Cancer cells, however, display higher levels of glycolysis followed by cytoplasmic fermentation, and reduced levels of oxidative phosphorylation. It was thought that increased glycolysis is associated with mitochondrial dysfunction, but how these phenomena are functionally linked was not known. Understanding how these processes are regulated will be essential for developing more effective anti-cancer therapies. Here, we show that induction of mitochondrial dysfunction by either genetic or chemical approaches results in a switch from oxidative phosphorylation to glycolysis. We further show that NADPH oxidase (NOX), an enzyme known to catalyze the oxidation of NAD(P)H, also plays a critical role in supporting increased glycolysis in cancer cells by generating NAD+, a substrate for one of the key glycolytic reactions. Inhibition of NOX leads to inhibition of cancer cell proliferation in vitro and suppression of tumor growth in vivo. This study reveals a novel function for NOX in cancer metabolism, explains the increased glycolysis observed in cancer cells, and identifies NOX as a potential anti-cancer therapeutic target.

The Role of Quantitative NPTX2 Hypermethylation as a Novel Serum Diagnostic Marker in Pancreatic Cancer

The majority of pancreatic cancers are found to be unresectable, and the only chance for cure lies on early detection and complete resection. Several genes have been discovered to be aberrantly methylated in primary pancreatic cancer tissue, and this cancer DNA can be detected in the plasma. The aims of this study were to develop a novel diagnostic marker based on epigenetic characteristics of pancreatic cancer. We enrolled 104 patients with pancreatic cancer, 60 with chronic pancreatitis, and 5 with benign biliary stone diseases. The blood samples were collected before surgery or any kinds of treatment modalities. DNA was extracted from the plasma of each patient, and NPTX2 (neuronal pentraxin II) CpG island hypermethylation was examined quantitatively by real-time polymerase chain reaction. NPTX2 hypermethylation levels were significantly higher compared with chronic pancreatitis (P = 0.016). The sensitivity and specificity were 80% and 76%, respectively (cutoff = 0.015). NPTX2 gene hypermethylation level was significantly elevated in correlation with higher American Joint Committee on Cancer stages. The aberrantly methylated NPTX2 gene may help to distinguish between chronic pancreatitis and pancreatic cancer with conventional diagnostic tools and could become a valuable diagnostic marker.

Novel diagnostic value of circulating miR-18a in plasma of patients with pancreatic cancer.

Background:Several recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in the plasma/serum. We hypothesised that miR-18a in the plasma is a potential biomarker in patients with pancreatic cancer.Methods:miR-18a is located in the miR-17–92 cluster and reported to be highly expressed in pancreatic cancer tissues. This study was divided into three parts: (1) Confirmation of higher miR-18a levels in primary pancreatic cancer tissues and cell lines than in normal pancreatic tissues and a human fibroblast cell line. (2) Evaluation of the plasma miR-18a assay using quantitative RT–PCR by comparing plasma results obtained from 36 patients with pancreatic cancer and from 30 healthy volunteers. (3) Evaluation of the assay for monitoring tumour dynamics in patients with pancreatic cancer.Results:(1) The expression of miR-18a was significantly higher in pancreatic cancer tissues (P=0.012) and pancreatic cancer cell lines (P=0.015) than in normal tissues and fibroblasts. (2) Plasma concentrations of miR-18a were significantly higher in pancreatic cancer patients than in controls (P<0.0001). The value of the area under the receiver-operating characteristic curve (AUC) was 0.9369. (3) Plasma levels of miR-18a were significantly lower in postoperative samples than in preoperative samples (P=0.0077).Conclusion:Circulating miR-18a might provide new complementary tumour markers for pancreatic cancer.

Nodal/Activin Signaling Drives Self-Renewal and Tumorigenicity of Pancreatic Cancer Stem Cells and Provides a Target for Combined Drug Therapy

Nodal and Activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-β. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.

Abstract B45: Embryogenesis meets tumorigenesis: Nodal/activin signaling drives self-renewal and invasiveness of pancreatic cancer stem cells

Abstract Nodal and activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and/or Activin regulate self-renewal and invasiveness of pancreatic cancer stem cells. While their expression was hardly detectable in differentiated pancreatic cancer cells, cancer stem cells demonstrate a drastic upregulation for Nodal and, to a lesser extent, Activin while TGF-β remained unchanged. Pancreatic stellate cells as the putative microenvironment for cancer stem cells produce very high amounts of Activin, and, to a lesser extent, Nodal. Knockdown and pharmacological inhibition of their common receptor Alk4/7 both significantly, but reversibly reduced cancer stem cell self-renewal / invasiveness and cancer stem cells subsequently became sensitive to gemcitabine. Importantly, while orthotopically engrafted primary human cancer cell suspensions were highly responsive to Nodal/Activin inhibition plus gemcitabine, engrafted primary human pancreatic cancer tissue xenografts containing massive stroma did not slow down tumor progression. Intriguingly, the addition of a stroma-targeting sonic hedgehog pathway inhibitor resulted in enhanced delivery of the Nodal/Activin inhibitor and gemcitabine, respectively, and translated into long-term disease stabilization. Therefore, modulation of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a novel therapeutic strategy for targeted elimination of cancer stem cells in order to overcome their resistance towards gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B45.

Clusterin confers gmcitabine resistance in pancreatic cancer

ObjectiveTo measure clusterin expression in pancreatic cancer tissues and cell lines and to evaluate whether clusterin confers resistance to gmcitabine in pancreatic cancer cells.MethodsImmunohistochemistry for clusterin was performed on 50 primary pancreatic cancer tissues and 25 matched backgrounds, and clusterin expression in 5 pancreatic cancer cell lines was quantified by Western blot and PT-PCR. The correlation between clusterin expression level and gmcitabine IC50 in pancreatic cancer cell lines was evaluated. The effect of an antisense oligonucleotide (ASO) against clusterin(OGX-011) on gmcitabine resistance was evaluated by MTT assays. Xenograft model was used to demonstrate tumor growth.ResultsPancreatic cancer tissues expressed significantly higher levels of clusterin than did normal pancreatic tissues (P < 0.01). Clusterin expression levels were correlated with gmcitabine resistance in pancreatic cancer cell lines, and OGX-011 significantly decreased BxPc-3 cells resistance to gmcitabine (P < 0.01). In vivo systemic administration of AS clusterin and gmcitabine significantly decreased the s.c. BxPC-3 tumor volume compared with mismatch control ODN plus gmcitabine.ConclusionOur finding that clusterin expression was significantly higher in pancreatic cancer than in normal pancreatic tissues suggests that clusterin may confer gmcitabine resistance in pancreatic cancer cells.

Abstract 2611: Inhibition of the immunoproteasome subunit LMP2 by UK-101 leads to apoptosis and cell cycle arrest in pancreatic cancer

Abstract Background: The immunoproteasome is an alternative form of the constitutive proteasome and it has been increasingly shown to play an important role in a number of diseases including cancer and inflammatory diseases. We have previously developed ‘UK-101’, a small molecule that selectively inhibits the immunoproteasome catalytic subunit LMP2. In our present study, we investigated the therapeutic potential of the immunoproteasome-targeting approach in pancreatic cancer using UK-101 as a lead compound. Methods: The expression of LMP2 was assessed in primary pancreatic cancer tissues and established pancreatic cancer cell lines via immunohistochemical (IHC) and immunoblotting analyses. Using pancreatic cancer cells expressing LMP2, we have investigated the effects of UK-101 (an LMP2 inhibitor), general proteasome inhibitors (bortezomib and carfilzomib) and gemcitabine on cell survival, apoptosis and cell cycle progression. We also examined the effects of UK-101 on various molecular markers associated with apoptosis and cell cycle control. Results: Our immunoblotting and IHC analyses indicated that the LMP2 levels are substantially upregulated in primary pancreatic cancer tissues as well as established pancreatic cancer cell lines compared to nonmalignant pancreatic tissues. The IHC staining on a tumor tissue array demonstrated positive immunostaining for LMP2 in approximately 53% of pancreatic cancer specimens evaluated (n=23 out of 43) while the normal pancreatic duct showed a negative LMP2 staining (n=11). Using Panc-1 (gemcitabine-resistant) and BxPc-3 (gemcitabine-sensitive) cells, we observed that the UK-101 treatment leads to a covalent modification of LMP2, a decreased proteasomal proteolytic activity as well as apoptotic cell death and cell cycle arrest. Conclusion: Our results show that LMP2 is upregulated in pancreatic cancer and that the targeted inhibition of LMP2 using UK-101 induces apoptotic cell death and cell cycle arrest. Taken together, these results suggest that the immunoproteasome may be a potential therapeutic target that can be exploited for pancreatic cancer where the existing therapy including gemcitabine shows limited efficacy. 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 2611. doi:10.1158/1538-7445.AM2011-2611

Enhanced cell migration and invasion of CD133+ pancreatic cancer cells cocultured with pancreatic stromal cells

Recently, cancer stem cells have been reported as a new therapeutic target in pancreatic cancer as well as other cancers, but the specific role of these cells is unknown. The authors investigated the functional roles of CD133+ cells, 1 of the putative cancer stem cell candidates in pancreatic cancer. CD133 expression was assessed in human pancreatic cancer and cancer cell lines by quantitative real-time reverse transcriptase polymerase chain reaction and flow cytometry. Next, they compared the ability of CD133+ and CD133- cells to proliferate, migrate, and invade using 2 pancreatic cancer cell lines. In particular, they evaluated the relationship between CD133+ cells and primary pancreatic stromal cells. CD133 was expressed in primary human pancreatic cancer tissues and some cancer cell lines, whereas there was little expression in primary normal pancreatic epithelial cells and primary pancreatic stromal cells. CD133+ cells, isolated by flow cytometry, showed increased cell proliferation under anchorage-independent conditions (P<.01), and enhanced migration and invasion, particularly when cocultured with primary pancreatic stromal cells (P<.001). Chemokine-related receptor-4 (CXCR4), markedly overexpressed in CD133+ cells, may be responsible for the increased invasive ability of the cells cocultured with pancreatic stromal cells, which express stromal derived factor-1, the ligand to CXCR4. These data suggest that CD133+ cells exhibit more aggressive behavior, such as increased cell proliferation, migration, and invasion, especially in the presence of pancreatic stromal cells. The targeting therapy for the interaction between CD133+ cancer cells and stromal cells may be a new approach for the treatment of pancreatic cancer.

Abstract 5295: S100A4 regulates tumor growth, motility, and invasion in pancreatic cancer

Abstract S100A4 protein belongs to the S100 subfamily, which has grown to be one of the large subfamilies of the EF-hand Ca2+-binding proteins, and overexpression of S100A4 is suggested to associate with cell proliferation, invasion, and metastasis. We observed frequent overexpression of S100A4 in pancreatic cancer cell lines by quantitative real-time RT-PCR and Western blotting. Tissue microarray analysis validated S100A4 overexpression in primary pancreatic cancer tissues, and overexpression of S100A4 associated with perineural invasion. We further analyzed RNAi-mediated knockdown to address the possibility of its use as a therapeutic target for pancreatic cancer. The specific knockdown of S100A4 strongly suppressed cell growth, induced G2 arrest and eventual apoptosis, and decreased cell migration. Furthermore, microarray analyses revealed that knockdown of S100A4 induced expression of the tumor suppressor genes PRDM2 and VASH1. We further established stably S100A4-expressing cell lines using two pancreatic cancer cell lines with low level of S100A4 expression; remarkable accelerated cell growth and motility was observed after induction of S100A4, whereas no change was observed in the control cells. Comparison of the expressional profiles after siRNA-mediated knockdown and expression vector-mediated induction is underway, and the results will also be demonstrated and discussed. 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 5295.

Abstract 3209: The WRN RecQ helicase acts as a tumor suppressor in pancreatic adenocarcinoma

Abstract The vast majority of pancreatic adenocarcinoma is diagnosed in the geriatric population, and genes involved in cellular senescence such as the WRN-encoded RecQ helicase/exonuclease may be involved in pancreatic oncogenesis. Accumulating evidence suggests that WRN is a tumor suppressor gene (TSG), as a variety of cancers including pancreatic adenocarcinoma are associated with its epigenetic silencing or loss-of-function mutations. We hypothesize that wild-type WRN protects pancreatic epithelia from malignant transformation by regulating cell cycle progression and cell growth. First, we characterized a pancreatic adenocarcinoma cell line established from a human cadaver with Werner's syndrome (WS-Panc). WS-Panc had two loss-of-function mutations of WRN, formed adherent colonies in vitro, and expressed the pancreatic ductal epithelial marker cytokeratin 7. No oncogenic mutations of the epidermal growth factor receptor (EGFR), K-Ras nor B-Raf were detected by polymerase chain reaction (PCR), and immunologic assays revealed over-expression of lysyl oxidase-like 2, p53, and ß-catenin in WS-Panc. Fluorescent in situ hybrdization showed that WS-Panc over-expresses EGFR. Cytogenetic analyses showed normal karyotype. Strong telomerase activity was detected in WS-Panc by the Telomere Repeat Amplification Protocol. To test the hypothesis that WRN is a TSG, we forced ectopic expression of wild-type WRN in WS-Panc and are analyzing the biological properties and molecular features of transfected cells. Consistent with an anti-proliferative effect, wild-type WRN mRNA levels are uniformly repressed in a panel of sporadic pancreatic cancer cell lines from the American Type Culture Collection as shown by quantitative real-time PCR. In summary, we provide evidence that WRN is a TSG in pancreatic epithelia using WS-Panc and other pancreatic cancer cell lines. WS-Panc is the first cancer cell line carrying WRN loss-of-function mutations that represents a unique translational resource to elucidate WRN in the neoplastic state. Ongoing investigation of WRN in primary pancreatic cancer tissue and tumor models in vivo are expected to elucidate mechanistic links between aging and pancreatic cancer for the purpose of developing efficacious interventional strategies. 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 3209.

Abstract 1605: A possible target key molecule for treatment of pancreatic cancer, screened as cancer-specific immortalization associated gene

Abstract [Background] Pancreatic caner shows a poor prognosis, and thus development of an efficient therapy is needed. Last year, we have reported that 13 genes were screened in relation to an event “cancer cell immortalization” by means of comprehensive gene expression analyses of normal, mortal and immortal cells and tissue specimen, and that these might be potent candidates for novel targets of anticancer chemotherapy. [Purpose] GTSE1 gene has been selected as one of the 13 genes that expressed at higher levels in immortal cancer cells, though its biological function relative to growth regulation of cancer cells are yet to be determined. To clarify its roles, we performed in vitro experiments to validate its possibility as a target key molecule for treatment of cancer, specifically pancreatic one. [Materials and Methods] Quantitative analyses of gene expression levels were conducted by real-time RT-PCR method using gene specific-primers and Universal Probe Library probes (Roche). Whole genome gene expression analysis was conducted with 4×44K Whole Human Genome Microarray (Agilent). Knockdown experiments were performed in various cancer cells using Stealth RNAi and RNAiMax reagent (Invitrogen). Cell proliferation was measured with MTT assay. Caspase activities were analyzed using ApoAlert Caspase Profiling Plate (Clontech). Cell cycle profile was obtained by measuring PI-stained cells with FACS “Calibur” (BD Biosciences). [Results] GTSE1 expressed at much higher levels in primary pancreatic and endometrial cancer tissue specimens as compared with matched normal ones: it expressed at higher levels in pancreatic, esophageal, and lung cancer cell lines than normal cells: higher expression levels of GTSE1 were observed in transformants of lung fibroblast TIG-1 cells with SV40 and TERT as compared with TIG-1 cells harboring TERT alone. Treatment with GTSE1 siRNA revealed a significant inhibition of growth of various cancer cells including paclitaxel-resistant ovarian cancer KFr13Tx, cisplatin-resistant lung cancer PC-9/CDDP, and gemcitabine-resistant pancreatic cancer MIAPaCa-2 cells. GTSE1 knockdown induced alteration of cell cycle profile and apoptosis of pancreatic cancer cells: it enhanced caspase 2 and 3 activities. Moreover, to seek for downstream genes, we performed microarray analysis of KLM-1, MIAPaCa-2 and DLD-1 cells treated with GTSE1 siRNA for 72 hrs: We found a gene A that showed less than 1/8 gene expression levels as compared with negative control ones in all of the tested cells by using microarray: This was confirmed by real-time RT-PCR. Furthermore, knockdown of the selected gene also altered cell cycle profile and apoptosis of pancreatic cancer cells. [Conclusion] These results indicate that GTSE1 may be involved in cancer-specific immortalization and may be useful as a molecular target for cancer therapy including pancreatic one. 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 1605.

AURKA is one of the downstream targets of MAPK1/ERK2 in pancreatic cancer

DUSP6/MKP-3, a specific inhibitor of MAPK1/ERK2, frequently loses its expression in primary pancreatic cancer tissues. This evidence suggests that constitutive activation of MAPK1 synergistically induced by frequent mutation of KRAS2 and the loss of function of DUSP6 plays key roles in pancreatic carcinogenesis and progression. By profiling of gene expressions associated with downregulation of MAPK1 induced by exogenous overexpression of DUSP6 in pancreatic cancer cells, we found that AURKA/STK15, the gene encoding Aurora-A kinase, which plays key roles in cellular mitosis, was among the downregulated genes along with its related genes, which included AURKB, TPX2 and CENPA. An association of expression and promoter activity of AURKA with MAPK activity was verified. Knockdown of ETS2 resulted in a reduction of AURKA expression. These results indicate that AURKA is a direct target of the MAPK pathway and that its overexpression in pancreatic cancer is induced by hyperactivation of the pathway, at least via ETS2.

Immortalizing the complexity of cancer metastasis: Genetic features of lethal metastatic pancreatic cancer obtained from rapid autopsy

The virtual lack of well-characterized metastatic pancreatic cancer tissues for study has limited systematic studies of the metastatic process of this deadly disease. To address this important issue, we have instituted a rapid autopsy protocol for the collection of high quality tissues from patients with metastatic pancreatic cancer, called the Gastrointestinal Cancer Rapid Medical Donation Program (GICRMDP). At the time of preparation of this manuscript, 20 patients with metastatic pancreatic cancer and one patient with metastatic colon cancer have undergone a rapid autopsy in association with the GICRMDP. The average time interval achieved for these 21 patients was 8.0 hours, with more than 500 individual samples of matched high quality primary and metastatic pancreatic cancer tissues, peritoneal/pleural fluid and blood obtained so far. For the first four patients in which the autopsy was performed in

Abrogation of DUSP6 by hypermethylation in human pancreatic cancer

Our previous study indicated that DUSP6/MKP-3/PYST1 could act as a tumor suppressor in human pancreatic cancer. DUSP6 was frequently underexpressed in primary pancreatic cancer tissues by an unknown mechanism. In this study, we demonstrated that hypermethylation of the expressional control region of DUSP6 could account for its abrogation in cultured human pancreatic cancer cells and in primary pancreatic cancer tissues. First, we checked intrinsic transcriptional expression levels of DUSP6 by a quantitative real time PCR assay in 16 cultured pancreatic cancer cell lines and found that the cells could be classified into four groups: very-low-level expression, low-level expression, high-level expression, and very-high-level expression. We observed restored expression of DUSP6 after treatment with 5-azacytidine and trichostatin A, a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, respectively, in cells with intrinsically very-low-level and low-level expression of DUSP6. Using a sodium-bisulfite-modification assay, we found that CpG sequences in intron 1 of DUSP6 were heavily methylated in MIA PaCa-2 and PAN07JCK, both showing the very low level of intrinsic expression of the gene. On the other hand, no methylation in this region was detected in 14 other cell lines. We checked the methylation state of this region by a methylation-specific PCR method in 12 primary pancreatic cancer tissues and compared it with the expression state of DUSP6 investigated by immunohistochemistry. Methylation was detected in five of eight cases with abolished expressions of DUSP6, four of which were poorly differentiated adenocarcinoma. On the other hand, none of the four cases with preserved expression of DUSP6 showed methylation. The methylation state significantly correlated with both the abolishment of protein expression (p = 0.038) and the histological subtype of adenocarcinoma (p = 0.023) by chi-square test. These results indicate that hypermethylation of the CpG islands in intron 1 may account for the strong suppression of DUSP6 expression. Other mechanism(s) and/or other CpG sites outside of our investigation may have some influence upon expressional suppression. Our combined results suggest that hypermethylation with modification of histone deacetylation play an important role in transcriptional suppression of DUSP6 in human pancreatic cancer.

Claudin 4 Protein Expression in Primary and Metastatic Pancreatic Cancer

We performed a comprehensive immunohistochemical evaluation of claudin 4 protein expression in paraffin-embedded tissue samples from 72 patients with primary infiltrating pancreatic cancer, 38 patients with metastatic pancreatic cancer, and a panel of normal control tissue samples from various organs. In 11 samples of primary infiltrating pancreatic cancer, foci of pancreatic intraepithelial neoplasia (PanIN) were present and also were analyzed for claudin 4 protein expression. Intense positive claudin 4 immunolabeling was noted within virtually all primary (71/72 [99%]) and metastatic (49/49 [100%]) pancreatic cancer tissue samples analyzed and in 10 of 11 samples of PanIN. In all cases, immunolabeling was noted in a membranous distribution. Claudin 4 protein also was detectable in normal breast, prostate, bladder, and gastrointestinal mucosa, although expression was substantially less intense than that seen in pancreatic cancer tissue samples. Our findings support the use of claudin 4 as a target for novel therapeutics or radioimaging of infiltrating pancreatic cancer. Furthermore, the finding of claudin 4 overexpression within pancreatic intraepithelial neoplasia, the precursor lesion of pancreatic cancer, suggests a potential benefit of imaging claudin 4 before the development of an invasive carcinoma.

Claudin 4 protein expression in primary and metastatic pancreatic cancer: support for use as a therapeutic target.

We performed a comprehensive immunohistochemical evaluation of claudin 4 protein expression in paraffin-embedded tissue samples from 72 patients with primary infiltrating pancreatic cancer, 38 patients with metastatic pancreatic cancer, and a panel of normal control tissue samples from various organs. In 11 samples of primary infiltrating pancreatic cancer, foci of pancreatic intraepithelial neoplasia (PanIN) were present and also were analyzed for claudin 4 protein expression. Intense positive claudin 4 immunolabeling was noted within virtually all primary (71/72 [99%]) and metastatic (49/49 [100%]) pancreatic cancer tissue samples analyzed and in 10 of 11 samples of PanIN. In all cases, immunolabeling was noted in a membranous distribution. Claudin 4 protein also was detectable in normal breast, prostate, bladder, and gastrointestinal mucosa, although expression was substantially less intense than that seen in pancreatic cancer tissue samples. Our findings support the use of claudin 4 as a target for novel therapeutics or radioimaging of infiltrating pancreatic cancer. Furthermore, the finding of claudin 4 overexpression within pancreatic intraepithelial neoplasia, the precursor lesion of pancreatic cancer, suggests a potential benefit of imaging claudin 4 before the development of an invasive carcinoma.

Potential Tumor Suppressive Pathway Involving DUSP6/MKP-3 in Pancreatic Cancer

We previously found frequent loss of heterozygosity at 12q21 and 12q22-q23.1 in primary pancreatic cancers, and the DUSP6/MKP-3 gene residing in this region at 12q22 lost its expression in the great majority of pancreatic cancer cell lines. The DUSP6/MKP-3 protein is a dual-specificity phosphatase that dephosphorylates the active form of ERK, making a feedback loop to control ERK activity. Gain-of-function mutations of KRAS2 occur in the great majority of pancreatic cancer cells, and loss of expression of DUSP6/MKP-3 may synergistically promote constitutive activation of ERK and uncontrolled cell growth. To study loss of the feedback pathway and its impact on pancreatic cancer cell growth, we first investigated the expression of DUSP6/MKP-3 in primary pancreatic cancer tissues immunohistochemically; we found up-regulation in mildly as well as severely dysplastic/in situ carcinoma cells and down-regulation in invasive carcinoma, especially in the poorly differentiated type. Adenovirus-mediated reintroduction of DUSP6/MKP-3 into cultured pancreatic cancer cells induced strong expression of recombinant DUSP6/MKP-3 and reduction of phosphorylated ERK in a dose-dependent manner based on the multiplicity of infection and resulted in suppression of cell growth. Moreover, analyses by flow cytometry and immunocytochemistry revealed that the exogenous expression of DUSP6/MKP-3 induced apoptosis. These results show that DUSP6 exerts apparent tumor-suppressive effects in vitro and suggest that DUSP6 is a strong candidate tumor suppressor gene at 12q22 locus.

Human BAC contig covering the deleted region in pancreatic cancer at 12q21.

In sporadic human primary pancreatic cancer tissues, loss of heterozygosity is frequently observed in the 1-cM region between D12S81 and D12S1719 at 12q21. Loss of this chromosome arm is known to be associate with a poor prognosis in pancreatic cancer patients. Herein we report a complete contig of human bacterial artificial chromosome (BAC) clones covering the deleted region. The region was covered by 21 BAC clones in a minimum tiling path. The clones were confirmed to exist at 12q21 by fluorescence in situ hybridization. We identified novel 40 sequence tagged sites and mapped 10 expressed sequence tags in this region. The BAC contig reported here provides an avenue for determining the complete nucleotide sequence and mining putative tumor suppressor genes in the deleted region of pancreatic cancer at 12q21.