Pancreatic Ductal Adenocarcinoma Cell Growth Research Articles

The oncogenic kinase Pim-1 is modulated by K-Ras signaling and mediates transformed growth and radioresistance in human pancreatic ductal adenocarcinoma cells

Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27Kip1. Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.

Involvement of the Tubulin Tyrosine Ligase-Like Family Member 4 Polyglutamylase in PELP1 Polyglutamylation and Chromatin Remodeling in Pancreatic Cancer Cells

Polyglutamylation is a new class of posttranslational modification in which glutamate side chains are formed in proteins, although its biological significance is not well known. Through our genome-wide gene expression profile analyses of pancreatic ductal adenocarcinoma (PDAC) cells, we identified the overexpression of tubulin tyrosine ligase-like family member 4 (TTLL4) in PDAC cells. Subsequent reverse transcription-PCR and Northern blot analyses confirmed its upregulation in several PDACs. TTLL4 belongs to the TTLL family which was reported to have polyglutamylase activity. Knockdown of TTLL4 by short hairpin RNA in PDAC cells attenuated the growth of PDAC cells and exogenous introduction of TTLL4 enhanced cell growth. We also found that TTLL4 expression was correlated with polyglutamylation levels of a glutamate stretch region of the proline, glutamate, and leucine-rich protein 1 (PELP1) that was shown to interact with various proteins such as histone H3, and was involved in several signaling pathways through its function as a scaffold protein. PELP1 polyglutamylation could influence its interaction with histone H3 and affect histone H3 acetylation. We also identified the interaction of PELP1 with LAS1L and SENP3, components of the MLL1-WDR5 supercomplex involving chromatin remodeling. Our findings imply that TTLL4 could play important roles in pancreatic carcinogenesis through its polyglutamylase activity and subsequent coordination of chromatin remodeling, and might be a good molecular candidate for the development of new therapeutic strategies for pancreatic cancer.

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.

An integrated humoral and cellular response is elicited in pancreatic cancer by α‐enolase, a novel pancreatic ductal adenocarcinoma‐associated antigen

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5-year survival rate. alpha-Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to alpha-enolase. The present work was intended to assess the ability of alpha-enolase to induce antigen-specific T cell responses. We show that alpha-enolase-pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN-gamma and lyse PDAC cells but not normal cells. In vivo, alpha-enolase-specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to alpha-enolase, the existence of alpha-enolase-specific T cells was also demonstrated. Taken as a whole, these results indicate that alpha-enolase elicits a PDAC-specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer.

Over‐expression of cysteine proteinase inhibitor cystatin 6 promotes pancreatic cancer growth

Pancreatic ductal adenocarcinoma (PDAC) shows the worst mortality among the common malignancies and development of novel therapies for PDAC through identification of good molecular targets is an urgent issue. Among dozens of over-expressing genes identified through our gene-expression profile analysis of PDAC cells, we here report CST6 (Cystatin 6 or E/M) as a candidate of molecular targets for PDAC treatment. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis confirmed over-expression of CST6 in PDAC cells, but no or limited expression of CST6 was observed in normal pancreas and other vital organs. Knock-down of endogenous CST6 expression by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in maintaining viability of PDAC cells. Concordantly, constitutive expression of CST6 in CST6-null cells promoted their growth in vitro and in vivo. Furthermore, the addition of mature recombinant CST6 in culture medium also promoted cell proliferation in a dose-dependent manner, whereas recombinant CST6 lacking its proteinase-inhibitor domain and its non-glycosylated form did not. Over-expression of CST6 inhibited the intracellular activity of cathepsin B, which is one of the putative substrates of CST6 proteinase inhibitor and can intracellularly function as a pro-apoptotic factor. These findings imply that CST6 is likely to involve in the proliferation and survival of pancreatic cancer probably through its proteinase inhibitory activity, and it is a promising molecular target for development of new therapeutic strategies for PDAC.

GABAB receptor is a novel drug target for pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death. Smoking, diabetes, and pancreatitis are risk factors. It has been shown that the growth of PDAC and pancreatic duct epithelial cells is regulated by beta-adrenoreceptors (beta-ARs). The activity of beta-ARs in the central nervous system is counteracted by gamma-aminobutyric acid (GABA) via GABA B receptor-mediated inhibition of adenylyl cyclase. The aim of the study was to investigate if GABA B R inhibits beta-AR signaling in PDAC and pancreatic duct epithelial cells, thus blocking driving forces of cancer progression, such as cell proliferation and cell migration. Intracellular cAMP was measured by immunoassays, DNA synthesis by BrdU incorporation assays, activation of ERK1/2 by ERK activation assays, and Western blots and metastatic potential by cell migration assays in the human PDAC cell lines PANC-1 and BXPC-3 and immortalized human pancreatic duct epithelial cells HPDE6-C7. The expression of norepinephrine, PKAR IIalpha, and GABA in PDAC microarrays was assessed by immunohistochemistry. RESULTS.: Stimulation of the GABA B R by GABA or baclofen inhibited isoproterenol-induced cAMP signaling below base levels. ERK1/2 activity in response to isoproterenol was blocked by GABA, an effect enhanced by transient overexpression of the GABA B R and abolished by GABA B R knockdown. DNA synthesis and cell migration were stimulated by isoproterenol, responses blocked by GABA and baclofen. Norepinephrine and PKAR IIalpha were overexpressed while GABA was underexpressed in human PDAC tissue arrays. The data suggest the stimulation of GABA B R signaling as a novel target for the treatment and prevention of pancreatic cancer.

γ-Aminobutyric Acid (GABA) Stimulates Pancreatic Cancer Growth through Overexpressing GABAA Receptor π Subunit

Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter in the mature central nervous system, and GABA/GABA receptors are also present in nonneural tissues, including cancer, but their precise function in nonneuronal or cancerous cells has thus far been poorly defined. Through the genome-wide cDNA microarray analysis of pancreatic ductal adenocarcinoma (PDAC) cells as well as subsequent reverse transcription-PCR and Northern blot analyses, we identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. We also found the expression of this peripheral type GABAA receptor subunit in few adult human organs. Knockdown of endogenous GABRP expression in PDAC cells by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in PDAC cell viability. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRP-expressing PDAC cells, but not GABRP-negative cells, and GABAA receptor antagonists inhibited this growth-promoting effect by GABA. The HEK293 cells constitutively expressing exogenous GABRP revealed the growth-promoting effect of GABA treatment. Furthermore, GABA treatment in GABRP-positive cells increased intracellular Ca2+ levels and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade. Clinical PDAC tissues contained a higher level of GABA than normal pancreas tissues due to the up-regulation of glutamate decarboxylase 1 expression, suggesting their autocrine/paracrine growth-promoting effect in PDACs. These findings imply that GABA and GABRP could play important roles in PDAC development and progression, and that this pathway can be a promising molecular target for the development of new therapeutic strategies for PDAC.

Alpha-enolase elicits specific pancreatic adenocarcinoma specific-T cell responses in healthy donors and in tumor patients (48.4)

Abstract We have demonstrated that sera from pancreatic ductal adenocarcinoma (PDAC) patients contain IgG autoantibodies to α-enolase, a glycolytic enzyme that also works as surface plasminogen receptor. As this implies that α-enolase elicits a B-cell dependent humoral response in vivo in PDAC patients, its ability to induce a T cell mediated response to PDAC was investigated. Here we show that α-enolase-pulsed dendritic cells were capable to specifically stimulate healthy autologous T cells to proliferate, secrete IFN-γ and lyse PDAC cells. In vivo, α-enolase-specific T cells completely inhibited the growth of PDAC cells in immunodeficient mice. In PDAC patients, and in particular in those with circulating IgG autoantibodies to α-enolase, the existence of memory T cells to α-enolase was also demonstrated. As a whole, these results indicate that α-enolase elicits a PDAC-specific humoral and cellular responses. The sum of its features makes α-enolase a promising candidate for new immunotherapeutic strategies in the cure of PDAC that lacks until now of efficacious therapies to associate the conventional ones.

Novel tumor marker REG4 detected in serum of patients with resectable pancreatic cancer and feasibility for antibody therapy targeting REG4

Pancreatic ductal adenocarcinoma (PDAC) shows the worst mortality rate among common malignancies, with a 5-year survival rate of only 4%, and the majority of PDAC patients are diagnosed at an advanced stage in which no effective therapy is available at present. Although the proportion of curable cases is still not so high, surgical resection of early stage PDAC is the only way to cure the disease. Hence, establishment of a screening strategy to detect early stage PDAC by novel serological markers is required urgently, and development of novel molecular therapies for PDAC treatment is also eagerly expected. We here report overexpression of REG4, a new member of the regenerating islet-derived (REG) family, in PDAC cells on the basis of genome-wide cDNA microarray analysis as well as reverse transcription-polymerase chain reaction and immunohistochemical analysis. We also detected significant elevation of REG4 in the serum of some patients with early-stage PDAC using our enzyme-linked immunosorbent assay system, indicating the possibility of REG4 as a new serological marker of PDAC. Furthermore, we found that knockdown of endogenous REG4 expression in PDAC cell lines with small interfering RNA caused a decrease in cell viability. Concordantly, addition of recombinant REG4 to the culture medium enhanced growth of a PDAC cell line in a dose-dependent manner. A monoclonal antibody against REG4 neutralized its growth-promoting effects and attenuated significantly the growth of PDAC cells. These findings indicate that REG4 is a promising tumor marker to screen early-stage PDAC, and also that neutralization of REG4 by the antibody may offer novel potential tools for the treatment of PDAC.

Down-regulation of RAB6KIFL/KIF20A, a Kinesin Involved with Membrane Trafficking of Discs Large Homologue 5, Can Attenuate Growth of Pancreatic Cancer Cell

To identify novel molecular targets for treatment of pancreatic ductal adenocarcinoma (PDAC), we generated precise gene expression profiles of PDACs on a genome-wide cDNA microarray after populations of tumor cells were purified by laser microdissection. Through functional analysis of genes that were transactivated in PDACs, we identified RAB6KIFL as a candidate for development of drugs to treat PDACs at the molecular level. Knockdown of endogenous RAB6KIFL expression in PDAC cell lines by small interfering RNA drastically attenuated growth of those cells, suggesting an essential role for the gene product in maintaining viability of PDAC cells. RAB6KIFL belongs to the kinesin superfamily of motor proteins, which have critical functions in trafficking of molecules and organelles. Proteomics analyses using a polyclonal anti-RAB6KIFL antibody identified one of the cargoes transported by RAB6KIFL as discs, large homologue 5 (DLG5), a scaffolding protein that may link the vinexin-beta-catenin complex at sites of cell-cell contact. Like RAB6KIFL, DLG5 was overexpressed in PDACs, and knockdown of endogenous DLG5 by small interfering RNA significantly suppressed the growth of PDAC cells as well. Decreased levels of endogenous RAB6KIFL in PDAC cells altered the subcellular localization of DLG5 from cytoplasmic membranes to cytoplasm. Our results imply that collaboration of RAB6KIFL and DLG5 is likely to be involved in pancreatic carcinogenesis. These molecules should be promising targets for development of new therapeutic strategies for PDACs.