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. [Cancer Res 2008;68(3):918-26]

A better understanding of drug resistance mechanisms is required to improve outcomes in patients with pancreatic cancer. Here we characterized patterns of sensitivity and resistance to three conventional chemotherapeutic agents with divergent mechanisms of action (gemcitabine, 5-fluorouracil, and cisplatin) in pancreatic cancer cells. Four (L3.6pl, BxPC-3, CFPAC-1, SU86.86) were sensitive and five (PANC-1, Hs766T, AsPC-1, MIAPaCa-2, Mpanc96) were resistant to all 3 agents based on GI50 (50% growth inhibition). Gene expression profiling and unsupervised hierarchical clustering revealed that the sensitive and resistant cells formed two distinct groups and differed in expression of specific genes including several features of “epithelial-mesenchymal transition” (EMT). Interestingly, an inverse correlation between E-cadherin and its transcriptional suppressor, Zeb-1 was observed in the gene expression data and was confirmed by real time PCR. Independent validation experiment using 5 new pancreatic cancer cell lines confirmed that an inverse correlation between E-cadherin and Zeb-1 correlated closely with resistance to gemcitabine, 5-fluorouracil, and cisplatin. Silencing Zeb-1 in the mesenchymal lines not only increased the expression of E-cadherin but also other epithelial markers such as EVA1 and MAL2 and restored drug sensitivity. Importantly, immunohistochemical analysis of E-cadherin and Zeb-1 in primary tumors confirmed that expression of the two proteins was mutually exclusive (p=0.012). Therefore, our results suggest that Zeb-1 and other regulators of EMT may maintain drug resistance in human pancreatic cancer cells, and therapeutic strategies to inhibit Zeb-1 and reverse EMT should be evaluated.

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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-B. 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.

The Kras gene is mutated to an oncogenic form in most pancreatic tumors. However, early attempts to use this molecule as a specific biomarker of the disease, or inhibit its activity as a cancer therapy, failed. This left a situation in which everyone was aware of the association between this important oncogene and pancreatic cancer, but no one knew what to do about it. Recent findings have changed this picture—many assumptions made about KRAS and its role in pancreatic cancer were found to be incorrect. Several factors have contributed to increased understanding of the activities of KRAS, including creation of genetically engineered mouse models, which have allowed for detailed analyses of pancreatic carcinogenesis in an intact animal with a competent immune system. Cancer genome sequencing projects have increased our understanding of the heterogeneity of individual tumors. We also have a better understanding of which oncogenes are important for tumor maintenance and are therefore called “drivers.” We review the advances and limitations of our knowledge about the role of Kras in development of pancreatic cancers and the important areas for future research.

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.cell proliferation ͉ tumor progression ͉ zinc uptake

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.

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH 2 -terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0 -10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90 -240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor ␣ receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor ␣ (TGF␣) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGF␣ cleavage 120 -180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGF␣. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGF␣. Neutralization of TGF␣ function by an anti-TGF␣ antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGF␣-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.© 1999 by The American Society for Cell Biology 2493 INTRODUCTIONIonizing radiation has been shown to activate multiple signaling pathways within cells in vitro, which can lead to either increased cell death or increased proliferation depending on the cell type, the radiation dose, and the culture conditions (Xia et al., 1995;Rosette and Karin, 1996;Santana et al., 1996;Chmura et al., 1997;Schmidt-Ullrich et al., 1997;Carter et al., 1998;Haimovitz-Friedman, 1998;Kavanagh et al., 1998). Recently, a novel cellular target for ionizing radiation has been shown to be the ...