Craig D. Logsdon

Cancer-Associated Stromal Fibroblasts Promote Pancreatic Tumor Progression

Pancreatic adenocarcinoma is characterized by a dense background of tumor associated stroma originating from abundant pancreatic stellate cells. The aim of this study was to determine the effect of human pancreatic stellate cells (HPSC) on pancreatic tumor progression. HPSCs were isolated from resected pancreatic adenocarcinoma samples and immortalized with telomerase and SV40 large T antigen. Effects of HPSC conditioned medium (HPSC-CM) on in vitro proliferation, migration, invasion, soft-agar colony formation, and survival in the presence of gemcitabine or radiation therapy were measured in two pancreatic cancer cell lines. The effects of HPSCs on tumors were examined in an orthotopic murine model of pancreatic cancer by co-injecting them with cancer cells and analyzing growth and metastasis. HPSC-CM dose-dependently increased BxPC3 and Panc1 tumor cell proliferation, migration, invasion, and colony formation. Furthermore, gemcitabine and radiation therapy were less effective in tumor cells treated with HPSC-CM. HPSC-CM activated the mitogen-activated protein kinase and Akt pathways in tumor cells. Co-injection of tumor cells with HPSCs in an orthotopic model resulted in increased primary tumor incidence, size, and metastasis, which corresponded with the proportion of HPSCs. HPSCs produce soluble factors that stimulate signaling pathways related to proliferation and survival of pancreatic cancer cells, and the presence of HPSCs in tumors increases the growth and metastasis of these cells. These data indicate that stellate cells have an important role in supporting and promoting pancreatic cancer. Identification of HPSC-derived factors may lead to novel stroma-targeted therapies for pancreatic cancer.

S100P Stimulates Cell Proliferation and Survival via Receptor for Activated Glycation End Products (RAGE)

S100P is a member of the S100 protein family that is expressed in several malignant neoplasms. Currently the effects of this molecule on cell function are unknown. In the present study we investigated the biological effects and mechanisms of action of S100P using NIH3T3 cells. Expression of S100P in NIH3T3 cells led to the presence of S100P in the culture medium, increased cellular proliferation, and enhanced survival after detachment from the culture substrate or after exposure to the chemotherapeutic agent 5-flurouracil. The proliferation and survival effects of S100P expression were duplicated in a time- and concentration-dependent manner by the extracellular addition of purified S100P to wild-type NIH3T3 cells and correlated with the activation of extracellular-regulated kinases (Erks) and NF-κB. To determine the mechanisms involved in these effects, we tested the hypothesis that S100P activated RAGE (receptor for activated glycation end products). We found that S100P co-immunoprecipitated with RAGE. Furthermore, the effects of S100P on cell signaling, proliferation, and survival were blocked by agents that interfere with RAGE including administration of an amphoterin-derived peptide known to antagonize RAGE activation, anti-RAGE antibodies, and by expression of a dominant negative RAGE. These data suggest that S100P can act in an autocrine manner via RAGE to stimulate cell proliferation and survival.

StellaTUM: current consensus and discussion on pancreatic stellate cell research

The field of pancreatic stellate cell (PSC) biology is very young, as the essential in-vitro tools to study these cells (ie, methods to isolate and culture PSC) were only developed as recently as in 1998. Nonetheless, there has been an exponential increase in research output in this field over the past decade, with numerous research groups around the world focusing their energies into elucidating the biology and function of these cells. It is now well established that PSC are responsible for producing the stromal reaction (fibrosis) of two major diseases of the pancreas—chronic pancreatitis and pancreatic cancer. Despite exponentially increasing data, the methods for studying PSC remain variable. Although within individual laboratories methods are consistent, different methodologies used by various research groups make it difficult to compare results and conclusions. This article is not a review article on the functions of PSC. Instead, members of the Pancreatic Star Alliance (http://www.pancreaticstaralliance.com) discuss here and consolidate current knowledge, to outline and delineate areas of consensus or otherwise (eg, with regard to methodological approaches) and, more importantly, to identify essential directions for future research. Hepatic stellate cells (HSC) were first described by Karl von Kupffer in 1876; however, similar cells in the pancreas were first observed in the 1980s.1–3 In 1998, Apte et al 4 and Bachem et al 5 isolated and cultured PSC.4 5 In the normal pancreas, PSC are located in close proximity to the basal aspect of pancreatic acinar cells. In sections immunostained for the marker desmin (a cytoskeletal protein), quiescent PSC can be seen as cells with a central cell body and long cytoplasmic projections extending along the base of adjacent acinar cells similar to that of pericytes in the mammary gland. …

Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis

BACKGROUND & AIMS The molecular mechanisms underlying pancreatitis are largely unknown. The goal of this study was to identify an early genetic event that correlated with pancreatitis. METHODS Differential display of messenger RNAs (mRNAs) was conducted on normal pancreas vs. those of animals with secretagogue-induced pancreatitis. Northern blots from normal animals and animals with experimental acute pancreatitis were probed with cloned complementary DNAs for chemokines. Pancreatitis was induced with cerulein and by retrograde injection of bile salts. Immunocytochemistry was used to identify the source of chemokine expression. Pyrrolidine dithiocarbamate was tested for effects on chemokine expression and pancreatitis. RESULTS A differentially amplified band was consistently observed early after cerulein hyperstimulation. This band was identified as a portion of the mob-1 gene, an alpha-chemokine. Northern analysis indicated that mRNAs for mob-1 and another chemokine, mcp-1, were induced after cerulein hyperstimulation in vivo. mob-1 mRNA was also induced by retrograde injection of bile salts and by cerulein in acinar cells in vitro. mob-1 protein was localized to exocrine cells in pancreata of diseased animals. Pyrrolidine dithiocarbamate inhibited both chemokine gene expression and early inflammatory characteristics of pancreatitis. CONCLUSIONS Chemokines are induced in acinar cells by treatments that induce pancreatitis and may play an important role in the early stages of the disease.

S100P Promotes Pancreatic Cancer Growth, Survival, and Invasion

Purpose: In the current study, we examined the functional significance and mechanism of action of S100P in pancreatic cancer cells. Experimental Design: S100P levels were increased in Panc-1 cells, which do not express S100P, by transfection with an S100P cDNA and S100P levels were reduced in BxPC3 cells, which express high levels of S100P, by small interfering RNA gene silencing. Effects of these manipulations on cell proliferation, resistance to apoptotic insults, cell migration, and invasion were estimated in vitro using standard assays. The influences of S100P on tumor growth in vivo were studied using xenograft mouse models. To identify the mechanisms involved in these responses, coimmunoprecipitation studies were conducted with S100P with receptor for advanced glycation end products (RAGE) and the effects of inhibiting RAGE using an antagonistic peptide were analyzed. Results: S100P levels correlated with the rates of cell proliferation, survival, migration, and invasion in both cell models in vitro. In vivo, increased S100P levels increased the growth of tumors in mice with s.c.-implanted Panc-1 cells and decreased S100P levels decreased tumor growth after orthotopic implantation of BxPC-3 cells. A direct interaction between S100P and RAGE was indicated by coimmunoprecipitation of these molecules from pancreatic cancer cells. A RAGE antagonist peptide inhibited this interaction and also inhibited the biological effects of S100P on these cells in vitro. Conclusions: These data suggest that S100P plays a major role in the aggressiveness of pancreatic cancer that is likely mediated by its ability to activate RAGE. Thus, interference with S100P may provide a novel approach for treatment of pancreatic cancer.

Silencing of the Hypoxia-Inducible Cell Death Protein BNIP3 in Pancreatic Cancer

Hypoxic conditions exist within pancreatic adenocarcinoma, yet pancreatic cancer cells survive and replicate within this environment. To understand the mechanisms involved in pancreatic cancer adaptation to hypoxia, we analyzed expression of a regulator of hypoxia-induced cell death, Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). We found that BNIP3 was down-regulated in nine of nine pancreatic adenocarcinomas compared with normal pancreas despite the up-regulation of other hypoxia-inducible genes, including glucose transporter-1 and insulin-like growth factor-binding protein 3. Also, BNIP3 expression was undetectable even after hypoxia treatment in six of seven pancreatic cancer cell lines. The BNIP3 promoter, which was remarkably activated by hypoxia, is located within a CpG island. The methylation status of CpG dinucleotides within the BNIP3 promoter was analyzed after bisulfite treatment by sequencing and methylation-specific PCR. Hypermethylation of the BNIP3 promoter was observed in all BNIP3-negative pancreatic cancer cell lines and eight of 10 pancreatic adenocarcinoma samples. Treatment of BNIP3-negative pancreatic cancer cell lines with a DNA methylation inhibitor, 5-aza-2′ deoxycytidine, restored hypoxia-induced BNIP3 expression. BNIP3 expression was also restored by introduction of a construct consisting of a full-length BNIP3 cDNA regulated by a cloned BNIP3 promoter. Restoration of BNIP3 expression rendered the pancreatic cancer cells notably more sensitive to hypoxia-induced cell death. In conclusion, down-regulation of BNIP3 by CpG methylation likely contributes to resistance to hypoxia-induced cell death in pancreatic cancer.

An NF-κB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.

Ras Activity Levels Control the Development of Pancreatic Diseases

BACKGROUND & AIMS Differentiated pancreatic acinar cells expressing endogenous levels of mutant K-Ras do not spontaneously develop pancreatic ductal adenocarcinoma (PDAC). However, we hypothesized that acinar cells would develop PDAC in the presence of Ras activity levels mimicking those of human tumor cells. METHODS We measured Ras activity in PDAC cells from mice and humans using a Raf pull-down assay. We compared the effects of acinar cell expression of mutant K-Ras at endogenous and elevated levels on Ras activity and on the development of PDAC. RESULTS Ras activity was greatly elevated in PDAC cells compared with nontransformed cells expressing endogenous levels of mutant K-Ras. Expression of endogenous levels of mutant K-Ras in differentiated acinar cells resulted in moderately elevated Ras activity and in sparse murine pancreatic intraepithelial neoplasias (mPanINs) that did not spontaneously advance to PDAC unless the tumor suppressor p53 was simultaneously deleted. In contrast, expression of mutant K-Ras at higher levels generated Ras activity equal to that in PDAC. High Ras activity mimicking levels in PDAC led to acinar cell senescence and generated inflammation and fibrosis resembling the histologic features of chronic pancreatitis. With higher Ras activity in acinar cells, abundant mPanINs formed and spontaneously progressed to both cystic papillary carcinoma and metastatic PDAC. CONCLUSIONS There is an important relationship between Ras activity levels and the progression of PDAC. Sufficient Ras activity in pancreatic acinar induces several important pancreatic disease manifestations not previously reported and supports a potential direct linkage between chronic pancreatitis, cystic papillary carcinoma, and PDAC.

Aberrant expression of zinc transporter ZIP4 (SLC39A4) significantly contributes to human pancreatic cancer pathogenesis and progression

Zinc is an essential trace element and catalytic/structural component used by many metalloenzymes and transcription factors. Recent studies indicate a possible correlation of zinc levels with the cancer risk; however, the exact role of zinc and zinc transporters in cancer progression is unknown. We have observed that a zinc transporter, ZIP4 (SLC39A4), was substantially overexpressed in 16 of 17 (94%) clinical pancreatic adenocarcinoma specimens compared with the surrounding normal tissues, and ZIP4 mRNA expression was significantly higher in human pancreatic cancer cells than human pancreatic ductal epithelium (HPDE) cells. This indicates that aberrant ZIP4 up-regulation may contribute to the pancreatic cancer pathogenesis and progression. We studied the effects of ZIP4 overexpression in pancreatic cancer cell proliferation in vitro and pancreatic cancer progression in vivo. We found that forced expression of ZIP4 increased intracellular zinc levels, increased cell proliferation by 2-fold in vitro, and significantly increased tumor volume by 13-fold in the nude mice model with s.c. xenograft compared with the control cells. In the orthotopic nude mice model, overexpression of ZIP4 not only increased the primary tumor weight (7.2-fold), it also increased the peritoneal dissemination and ascites incidence. Moreover, increased cell proliferation and higher zinc content were also observed in the tumor tissues that overexpressed ZIP4. These data reveal an important outcome of aberrant ZIP4 expression in contributing to pancreatic cancer pathogenesis and progression. It may suggest a therapeutic strategy whereby ZIP4 is targeted to control pancreatic cancer growth.

RAGE and RAGE Ligands in Cancer

The receptor for advanced glycation end-products (RAGE) is a multifunctional receptor with multiple ligands that is known to play a key role in several diseases, including diabetes, arthritis, and Alzheimers disease. Recent evidence indicates that this receptor also has an important role in cancer. RAGE ligands, which include the S100/calgranulins and high-mobility group box 1 (HMGB1) ligands, are expressed and secreted by cancer cells and are associated with increased metastasis and poorer outcomes in a wide variety of tumors. These ligands can interact in an autocrine manner to directly activate cancer cells and stimulate proliferation, invasion, chemoresistance, and metastasis. RAGE ligands derived from cancer cells can also influence a variety of important cell types within the tumor microenvironment, including fibroblasts, leukocytes, and vascular cells, leading to increased fibrosis, inflammation, and angiogenesis. Several of the cells in the tumor microenvironment also produce RAGE ligands. Most of the cancer-promoting effects of RAGE ligands are the result of their interaction with RAGE. However, these ligands also often have separate intracellular roles, and some may interact with other extracellular targets, so it is not currently possible to assign all of their effects to RAGE activation. Despite these complications, the bulk of the evidence supports the premise that the ligand-RAGE axis is an important target for therapeutic intervention in cancer.