Pancreatic Cancer Gene Research Articles

Netting Pancreatic Cancer Genes

A rare but deadly form of human pancreatic cancer harbors mutations in chromatin remodeling genes.

Netting Pancreatic Cancer Genes

Pancreatic neuroendocrine tumors (PanNETs) are aggressive human cancers that often develop silently and progress to untreatable metastatic disease prior to diagnosis. Using an exome sequencing strategy to identify recurrent somatic mutations in PanNETs, Jiao et al. (p. [1199][1], published online 20

Epigenetic Silencing of Tumor Suppressor Genes in Pancreatic Cancer

Without any alteration of DNA sequence, heritable changes in gene expression, caused by epigenetic pathways, are gaining a spotlight in research of diseases, and in particular, cancer. Although the dominant paradigm in cancer research, proposed by Vogelstein, suggested that cancer progression was caused by a sequential accumulation of genetic aberrations, basic science studies in epigenetics have now advanced our knowledge enough to apply its concepts and methodology to the study of cancer. In fact, chromatin dynamics and small RNAs are altered far more prevalently in cancer than genetic alterations and most important, can be reversible, lending themselves as attractive therapeutic targets. In the current review, the inactivation of p16 will be utilized as the most prominent example of epigenetic silencing of a tumor suppressor gene in pancreatic cancer. In addition, fundamental insight will be given into why and how epigenetics can be targeted for therapeutic purposes. This knowledge will help the reader in determining the breadth and depth of this field of study with potentially high impact to oncology.

Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

Tumor-specific Expression And Alternative Splicing Of The COL6A3 Gene in Pancreatic Cancer

Tumor-specific Expression And Alternative Splicing Of The COL6A3 Gene in Pancreatic Cancer

Screening Technologies for Target Identification in Pancreatic Cancer

Pancreatic cancer exhibits an extraordinarily high level of resistance to almost any kind of systemic therapy evaluated in clinical trials so far. Therefore, the identification of novel therapeutic targets is urgently required. High-throughput screens have emerged as an important tool to identify putative targets for diagnosis and therapy in an unbiased manner. More than a decade ago, microarray technology was introduced to identify differentially expressed genes in pancreatic cancer as compared to normal pancreas, chronic pancreatitis and other cancer types located in close proximity to the pancreas. In addition, proteomic screens have facilitated the identification of differentially secreted proteins in body fluids of pancreatic cancer patients, serving as possible biomarkers. Recently, RNA interference-based loss-of-function screens have been used to identify functionally relevant genes, whose knock-down has impact on pancreatic cancer cell viability, thereby representing potential new targets for therapeutic intervention. This review summarizes recent results of transcriptional, proteomic and functional screens in pancreatic cancer and discusses potentials and limitations of the respective technologies as well as their impact on future therapeutic developments.

Abstract ED02-03: Gene, environment, and risk factor interaction in pancreatic cancer

Abstract ED02-03: Gene, environment, and risk factor interaction in pancreatic cancer

MiRNA-96 Suppresses KRAS and Functions as a Tumor Suppressor Gene in Pancreatic Cancer

Therapeutic applications of microRNA (miRNA) in KRAS-driven pancreatic cancers might be valuable, but few studies have explored this area. Here, we report that miR-96 directly targets the KRAS oncogene and functions as a tumor-suppressing miRNA in pancreatic cancer cells. Ectopic expression of miR-96 through a synthetic miRNA precursor inhibited KRAS, dampened Akt signaling, and triggered apoptosis in cells. In human clinical specimens, miR-96 was downregulated or deleted where an association with KRAS elevations was observed. In vitro and in vivo assays established that miR-96 decreased cancer cell invasion and migration and slowed tumor growth in a manner associated with KRAS downregulation. Our findings identify miR-96 as a potent regulator of KRAS, which may provide a novel therapeutic strategy for treatment of pancreatic cancer and other KRAS-driven cancers.

Association of aldehyde dehydrogenase 2 gene polymorphism with pancreatic cancer but not colon cancer

Most of the acetaldehyde, a recognized animal carcinogen, generated during alcohol metabolism is eliminated by liver mitochondrial aldehyde dehydrogenase 2 (ALDH2). More than 40% of Japanese people have the inactive form of ALDH2, and inactive ALDH2 is a risk factor for multiple cancer of the esophagus, as well as head and neck cancer. Possible associations between pancreatic cancer and ALDH2 gene polymorphism, as well as between colon cancer and ALDH2 gene polymorphism, in conjunction with smoking and/or drinking habits, were examined in a Japanese population. Patients with pancreatic cancer (n = 187) and with colon cancer (n = 49) were examined. The drinking (5 g ethanol consumption/day) and/or smoking habits as well as ALDH2 gene polymorphism were examined. The age-matched control subjects were recruited in the NILS Longitudinal Study of Aging (LSA). Aging, smoking and inactive ALDH2, but not alcohol, are independent risk factors for pancreatic cancer. The frequency of smoking habits tended to be higher in patients with colon cancer compared with the patients without cancer. However, age, body mass index or the distribution of ALDH2 genotypes did not differ significantly among the patients with colon cancer, colon polyps and others. Inactive ALDH2 is an independent risk factor for pancreatic cancer, but inactive ALDH2 might not be a risk for colon cancer.

Mutated genes in pancreatic cancer

Pancreatic cancer continues to be a deadly malignancy with still high mortality and poor survival. Little progress has been made on the treatment of advanced pancreatic cancer despite the significant advances in understanding, diagnosis, and access to conventional and novel treatments. Molecular pathology of the lesion is the key of our understanding of the mechanisms underlying the development of this cancer and will probably help us in earlier diagnosis and better therapeutic results. New treatment strategies and a more careful evaluation of innovative therapies are clearly needed for this disease. In view of many findings pancreatic cancer should be regarded as a systemic disease, and over the last several years, investigators have gained a better understanding of the molecular biology and events that lead to the development of malignancies. We here review the novel developments in the systemic exploring for commonly mutated genes in pancreatic cancer.

46 An integrative structural and functional approach to pancreatic cancer gene discovery

Studies have shown that some patients with left-hemispheric brain tumors have an increased propensity for developing right-sided language support. However, the precise trigger for establishing co-dominant language function in brain tumor patients remains unknown. We analyzed the MR scans of patients with left-hemispheric tumors and either co-dominant (n = 35) or left-hemisphere dominant (n = 35) language function on fMRI to investigate anatomical factors influencing hemispheric language dominance. Of eleven neuroanatomical areas evaluated for tumor involvement, the basal ganglia was significantly correlated with co-dominant language function (p < 0.001). Moreover, among patients whose tumors invaded the basal ganglia, those with language co-dominance performed significantly better on the Boston Naming Test, a clinical measure of aphasia, compared to their left-lateralized counterparts (56.5 versus 36.5, p = 0.025). While further studies are needed to elucidate the role of the basal ganglia in establishing co-dominance, our results suggest that reactive co-dominance may afford a behavioral advantage to patients with left-hemispheric tumors.

Abstract 248: Functional characterization of MED29 gene in pancreatic cancer

Abstract Pancreatic cancer is a highly aggressive malignancy characterized by rapid progression and an extremely poor prognosis. More knowledge is needed to better understand the mechanisms of the disease pathogenesis. Identification of new disease genes can improve our understanding about the biology of pancreatic cancer and might provide us tools for improving both diagnostics and treatment. Previously, we performed a detailed characterization of the 19q13 locus, one the recurrently amplified regions in pancreatic cancer, and identified a set of putative amplification target genes that showed consistent overexpression in amplified cell lines versus non-amplified cell lines. Subsequent high-throughput RNAi screen targeting genes within the amplified region revealed a number of genes whose downregulation led to decreased cell viability in the amplified but not in the non-amplified cells. Of these, the mediator complex subunit 29 (MED29) was consistently overexpressed in all amplified cell lines and had the most dramatic effect on cell viability. By flow cytometry analysis, we showed that MED29 silencing resulted in G0/G1 cell cycle arrest and increased apoptosis in the amplified PANC-1 cells. MED29 is a subunit of a large multiprotein complex that is involved in transcriptional regulation and may function both as a transcriptional repressor and enhancer. To further define the function of MED29 we constructed a lentiviral vector for MED29 gene delivery. Mouse embryonic fibroblasts (NIH/3T3) and two pancreatic cancer cell lines with low basal MED29 expression (MIAPaCa2 and Hs700T) were transduced with a lentivirus vector containing MED29. Stable MED29 expression was confirmed in all three cell lines by quantitative RT-PCR and western blot analysis. MED29 expression led to statistically significant changes in proliferation and/or survival in both the NIH/3T3 cells and the pancreatic cancer cells. Finally, MED29 overexpressing human pancreatic cancer cell lines were subcutaneously inoculated into immunocompromized mice to follow tumor growth in vivo. Significant growth difference was observed between MED29 expressing cancer cells compared to control cells when injected into mice. To conclude, our results implicate MED29 as an important regulator of growth and survival in pancreatic cancer both in vivo and in vitro. 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 248.

Abstract 179: Identification of novel targets for aberrant methylation in pancreatic cancer by a newly developed method “methyl-CpG targeted transcriptional activation (MeTA)”

Abstract Identification and characterization of genes selectively hypermethylated in cancer may improve our understanding of the role of epigenetic alterations in tumorigenesis. In addition, genes frequently methylated in a tumor-specific manner could be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2’-deoxycytidine have been widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting methylation-mediated silenced genes in cancer and successfully developed a new method termed “methyl-CpG targeted transcriptional activation (MeTA)” by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD). Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the preliminary experiments using human embryonic kidney cell line 293T, we found that the combination of MeTA with gene expression microarray is useful; genes containing CpG islands can be efficiently identified (93.5% by MeTA vs. 66.8% by 5-aza-cytidine) even those that are difficult to find by the conventional DNA methylation inhibitors. We applied this method to twelve typical pancreatic cancer cell lines with a normal pancreatic ductal epithelial cell line HPDE as the control, and have searched for genes commonly upregulated after MeTA. Thirty-one genes were upregulated by two-folds or more in six pancreatic cancer cell lines or more. Among these, 26 genes have not been reported previously using the conventional DNA methylation inhibitors. Methylation statuses of three genes, NEFL, NEFM, and IRX4 were further examined by methylation-specific PCR and found that these three genes were aberrantly methylated in all of the analyzed tumors, 11 resected primary pancreatic cancer specimens and 22 pancreatic cancer cell lines. Our results suggest that “MeTA” is a highly efficient method to identify methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer. 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 179.

Enhanced Pancreatic Cancer Gene Therapy by Combination of Adenoviral Vector Expressing c-erb-B2 (Her-2/neu)-Targeted Immunotoxin with a Replication-Competent Adenovirus or Etoposide

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, and even under optimal therapy these patients face a poor prognosis. Here we report a novel gene therapy-based strategy to battle this disease. We show that the majority of pancreatic tumors overexpress c-erb-B2, which therefore might serve as a target for novel therapies. On the basis of these findings, we developed an adenoviral vector [Ad-e23(scFv)-PE40] encoding a c-erb-B2 (Her-2/neu)-targeted immunotoxin. To improve viral gene delivery we coinfected the therapeutic adenovirus with a replication-competent adenovirus (RCAd) at low doses that enhanced the transduction efficiency of the former virus. In addition, we show that target gene expression can be enhanced by adding etoposide (VP16) at nontherapeutic doses. To investigate the therapeutic efficacy of our approach we established a mouse model for advanced pancreatic cancer disease by intraperitoneal injection of pancreatic cancer cell lines, resulting in multifocal peritoneal xenograft tumors. Administration of Ad-e23(scFv)-PE40 in combination with RCAd and VP16 significantly inhibited tumor growth in mice, with no apparent systemic toxicity. In this study we show that c-erb-B2 might be an effective molecular target in the treatment of pancreatic tumors and that coadministration of a therapeutic c-erb-B2-targeted, non-replication-competent adenovirus with an RCAd and VP16 could be a powerful approach to effectively deliver therapeutic genes to tumors. As demonstrated, this strategy can be employed to effectively treat pancreatic cancer in particular, but may be modified to treat other types of cancer as well.

Gene discovery for complex diseases using exomic sequencing: identifying pancreatic cancer susceptibility genes

Traditional methods of gene discovery, candidate gene and linkage approaches, have yielded very few pancreatic cancer susceptibility loci, such that genetic basis of ~90% of the familial clustering of pancreatic cancer is unknown. The recent advances and cost reductions in genome sequencing have enabled us to identify pancreatic cancer susceptibility genes through exomic sequencing patients with a family history of pancreatic cancer. Individuals that have an inherited tumor suppressor gene often lose the wild- type allele through loss of heterozygosity or somatic mutation. Therefore, we examined a patient's tumor and germ-line DNA for genes with evidence of both inherited and somatic variants that were likely to result in the absence of a functional protein in the patient's tumor. We also analyzed data on normal genetic variation in published databases and in individuals without pancreatic cancer. These analyses identified PALB2 as candidate pancreatic cancer gene. The role of PALB2 in familial pancreatic cancer was validated by studying an additional 96 pancreatic cancer patients. Large-scale follow-up studies of over 500 familial pancreatic cancer patients are currently underway.

Functional analysis of LOXL2 in pancreatic carcinoma

Lysyl oxidase-like 2 (LOXL2) plays a part in epithelial-mesenchymal transition (EMT) by stabilizing the transcription factor SNAI1. Previous studies showed that LOXL2 is one of the most highly and specifically upregulated genes in pancreatic cancer. LOXL2 was also found to be strongly upregulated in the secretome of established pancreatic cancer cell lines. To get more insight into the aggressive growth and infiltrating nature of pancreatic cancer, we evaluated the functional role of LOXL2 in pancreatic cancer cells. Gene inhibition by small interfering RNAs was used to silence LOXL2 in pancreatic cancer cell lines MiaPaCa-2 and Panc1. Cell death, proliferation, and morphology of transfected cells were determined. Cell characteristics under cell stress and gemcitabine treatment were analyzed. Gene expression analysis of transfected cells by DNA microarray was used to understand the processes of chemosensitization. Silencing of LOXL2 in pancreatic cancer cells resulted in an augmented sensitivity toward gemcitabine treatment, with significantly elevated cell death and reduced viable cells. However, transfection had no direct effect on morphology or growth pattern of Mia PaCa-2 and Panc1 cell lines. Gene expression analysis identified among others the transcription factor E2F5 as possible target of LOXL2. Gene inhibition of the EMT regulatory gene LOXL2 resulted in a distinct sensitization toward gemcitabine. Additionally, gene expression analysis showed a role for LOXL2 in the regulation of different transcription factors associated with invasion and metastasis. Our results suggest that the improved response toward chemotherapy in LOLX2-silenced pancreatic cancer cells is possibly mediated by the transcription factor E2F5.

Using lentiviral vectors for efficient pancreatic cancer gene therapy

Pancreatic cancer (PC) remains a life-threatening disease. Efficient therapeutic gene delivery to PC-derived cells continues to present challenges. We used self-inactivated lentiviral vectors to transduce PC-derived cells in vitro and in vivo. We showed that lentiviral vectors transduce PC-derived cell lines with high efficiency (>90%), regardless of the differentiation state of the cell. Next, we transferred human interferon beta (hIFN-beta) gene. Expression of hIFN-beta in PC cells using lentiviral vectors resulted in the inhibition of cell proliferation and the induction of cell death by apoptosis. In vivo, lentiviral administration of hIFN-beta prevented PC tumor progression for up to 15 days following gene therapy, and induced tumor regression/stabilization in 50% of the mice treated. Again, hIFN-beta expression resulted in cancer cell proliferation inhibition and apoptosis induction. We provide evidence that human immunodeficiency virus (HIV)-1-based lentiviral vectors are very efficient for gene transfer in PC-derived cells in vitro and in vivo. As a consequence, delivery of hIFN-beta stopped PC tumor progression. Thus, our approach could be applied to the 85% of PC patients with a locally advanced disease.

Confirmation of DNA Microarray-Derived Differentially Expressed Genes in Pancreatic Cancer Using Quantitative RT-PCR

The fact that pancreatic ductal adenocarcinoma (PDAC) is still an exceptionally lethal disease with an annual mortality almost equivalent to its annual incidence has stimulated intense research efforts directed at understanding the underlying molecular mechanisms. By enabling simultaneous expression analysis of thousands of genes, microarray technology has significantly contributed to illuminating the pathophysiology of PDAC. Gene expression profiling studies have been performed for molecular classification of clinically relevant tumor subtypes and have shed light on various signaling pathways associated with tumor progression. Altered expression levels of several genes have been identified as correlating with functional in vitro data as well as patient survival, indicating the potential clinical value of transcrip-tional profiling. However, broad clinical use of array techniques for patient characterization has been hampered by their cost intensity and by limited inter-study comparability. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), as the most sensitive technique for mRNA detection and quantification, will complement arrays for the confirmation of individual transcripts in larger sample cohorts. This review highlights recent studies that addressed gene expression analysis with both methodologies and that identified components of theTGF-β signaling pathway, BNIP3, or periostin to be differentially expressed in PDAC. These studies demonstrated that the combination of microarray and RT-PCR technologies is a highly efficient and reliable approach for the identification of clinically important diagnostic and prognostic biomarkers, as well as for the discovery of novel therapeutic target candidates.

Construction of lentivirus carrying novel human pancreatic cancer gene S100P and green fluorescent protein gene

Objective:To construct lentivirus carrying both novel human pancreatic cancer gene S100P and green fluorescent protein (GFP) gene.Methods: The fragments containing all the exons of S100P were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP. The lentivirus was packaged and was used to transfect 293T cells together with pShuttle. The supernatant of virus-producing cells was harvested, concentrated, identified, and was used to infect 293T cells and pancreatic cancer cells. Fluorescent microscopy was used to observe the fluorescence in the 293T cells; and real time-PCR was used to examine the relative contents of S100P in pancreatic cancer cells.Results: Electrophoresis showed that the sequence of the RT-PCR product was consistent with the data of NCBI by DNA sequencing analysis. The lentivirus effectively transfected 293T cells. Strong green fluorescence was observed by fluorescent microscopy. The supernatant of lentivirus-transfected 293T cells effectively infected 293T cells and the relative content of S100P in the transfected pancreatic cancer cells was higher than that of control group.Conclusion: The lentivirus vector containing S100P-GFP recombinant gene have been successfully constructed, which provides a basis for further study of S100P function.

Polymorphic Variants in Hereditary Pancreatic Cancer Genes Are Not Associated with Pancreatic Cancer Risk

Inherited risk of pancreatic cancer has been associated with mutations in several genes, including BRCA2, CDKN2A (p16), PRSS1, and PALB2. We hypothesized that common variants in these genes, single nucleotide polymorphisms (SNP), may also influence risk for pancreatic cancer development. A clinic-based case-control study in non-Hispanic white persons compared 1,143 patients with pancreatic adenocarcinoma with 1,097 healthy controls. Twenty-eight genes directly and indirectly involved in the Fanconi/BRCA pathway (includes BRCA1, BRCA2, and PALB2) were identified and 248 tag SNPs were selected. In addition, 11 SNPs in CDKN2A, PRSS1, and PRSS2 were selected. Association studies were done at the gene level by principal components analysis, whereas recursive partitioning analysis was used to investigate pathway effects. At the individual SNP level, adjusted additive, dominant, and recessive models were investigated, and gene-environment interactions were also assessed. Gene level analyses showed no significant association of any genes with altered pancreatic cancer risk. Multiple single SNP analyses showed associations, which will require replication. Exploratory pathway analyses by recursive partitioning showed no association between SNPs and risk for pancreatic cancer. In a candidate gene and pathway SNP association study analysis, common variations in the Fanconi/BRCA pathway and other candidate familial pancreatic cancer genes are not associated with risk for pancreatic cancer.