Phenotype Of Pancreatic Cancer Cells Research Articles

Over‐expression of FoxM1 leads to epithelial–mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b, and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein.

RETRACTED: Notch-1 induces epithelial–mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes-1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment.

Effect of nicotine on chemoresistant phenotype as mediated through Src-dependent Id1 expression in pancreatic adenocarcinoma cells.

216 Background: The signaling pathways contributing to DNA-binding protein inhibitor Id1 expression and chemoresistance in pancreatic cancer remain unknown. Id1 plays a role in pancreatic tumor progression with tumor-promoting effects of nicotine regulating protein tyrosine kinase Src activation and Id1 expression, both associated with chemoresistance in other systems. We hypothesize Id1 expression regulates chemoresistance in pancreatic cancer through a nicotine-promoting Src-dependent pathway. Methods: We probed pancreatic cancer cell lines (L3.6pl, PANC-1, Mia-PaCa-2) for innate gemcitabine chemoresistance with cell viability MTT assay and western blot analysis of PARP cleavage programmed cell death. Gemcitabine-sensitive cells were exposed to rising gemcitabine concentrations to establish a resistant subtype, L3.6plGemRes. Protein analysis and mRNA expression were determined by western blot analysis and RT-PCR respectively. Induction of Src phosphorylation or Id1 expression was performed with nicotine (1 μM). Results: Inhibition of c-Src expression was performed with short-interfering RNA (siRNA). Nicotine-induced Src phosphorylation and Id1 expression. Inhibition of Src by siRNA resulted in decreased nicotine-induced Id1 expression. Inhibition of Src and Id1 expression by siRNA in innate or established gemcitabine resistant pancreatic cancer cells resulted in gemcitabine sensitization. To determine if nicotine contributes to gemcitabine chemoresistance, we exposed gemcitabine-sensitive cells to nicotine with subsequent exposure to gemcitabine IC50, 250 ng/ml, and cell viability assays confirmed a 2-fold increase in cell prolilferation and a 4.5-fold reduction in apoptosis. Further, nicotine induced phosphorylation of key signaling enzymes involved in proliferation and apoptosis, Erk1/2 and Akt respectively. Conclusions: In summary, we demonstrate that Id1, through a nicotine-promoting Src-dependent pathway, is necessary for establishment of a chemoresistant phenotype in pancreatic cancer cells. Understanding the signaling pathways involved in pancreatic tumor chemoresistance will lead to therapies resulting in improved tumor responses. No significant financial relationships to disclose.

Inhibition of Ankyrin-B Expression Reduces Growth and Invasion of Human Pancreatic Ductal Adenocarcinoma

Background: In spite of the increasing knowledge of the molecular pathology of pancreatic ductal adenocarcinoma (PDAC), treatment of this tumor still remains an unresolved problem. Thus, the identification of ‘novel’ genes involved in pancreatic tumor progression is essential for early diagnosis and new treatment regimens of PDAC. Ankyrin-B (ANK2) was identified as being overexpressed in PDAC in a previous study by our group. ANK2 overexpression has been described in several tumors; however, the function of ANK2 in pancreatic carcinoma has not been elucidated. Materials and Methods: In the present study, we confirmed ANK2 overexpression in PDAC and analyzed the effects of ANK2 knockdown in the pancreatic tumor cell line PANC-1. Results: ANK2 silencing reduced the activity of FAK, ERK1/2 and p38. Decreased ANK2 expression restrained migration and invasive potential of PANC-1 cells. Moreover, silencing of ANK2 decreased the proliferation of the pancreatic tumor cells and reduced their tumorigenicity in vitro and in vivo. Conclusion: Our results demonstrate that silencing of ANK2 expression reduced the malignant phenotype of pancreatic cancer cells, indicating that ANK2 represents a potential target for therapy of pancreatic cancer.

ZEB1 in Pancreatic Cancer

Pancreatic cancer is one of the most malignant human neoplasias. On the molecular level, epithelial-mesenchymal transition (EMT) has been demonstrated to contribute to the malignant phenotype of pancreatic cancer cells. ZEB1 is a transcriptional repressor that has been identified as an inducer of EMT. A negative feedback loop between ZEB1 and microRNA-200c has been shown to regulate this EMT induction in various models. With respect to pancreatic cancer, primary effects of EMT comprise increased local and distant tumor cell dissemination. Another recently described feature of the EMT is the acquisition of cancer stem cell traits. For pancreatic cancer cells, antagonism between ZEB1 and stemness-inhibiting micro-RNAs has been demonstrated to contribute to this process, providing experimental support for the migrating cancer stem cell (MCSC) hypothesis. ZEB1 has also been shown to be associated with drug resistance of pancreatic cancer cells. This article reviews the biological functions of ZEB1 with a focus on pancreatic cancer.

NF-κB promotes epithelial–mesenchymal transition, migration and invasion of pancreatic carcinoma cells

The transcription factor NF-κB is constitutively active in pancreatic adenocarcinoma. Here we explore the contribution of NF-κB to the malignant phenotype of pancreatic cancer cells in addition to its anti-apoptotic role. Block of NF-κB signalling by non-destructible IκBα rendered cells resistant to TGF-β-induced epithelial–mesenchymal transition (EMT). In contrast, NF-κB activation by TNF-α or expression of constitutively active IKK2 induced an EMT-phenotype with up-regulation of vimentin and ZEB1, and down-regulation of E-cadherin. EMT could also be induced in cells with defective TGF-β signalling. Functional assays demonstrated reduced or strongly enhanced migration and invasion upon NF-κB inhibition or activation, respectively.

Desmoplasia of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and is characterized by remarkable desmoplasia. The desmoplasia is composed of extracellular matrix (ECM) proteins, myofibroblastic pancreatic stellate cells, and immune cells associated with a multitude of cytokines, growth factors, and ECM metabolizing enzymes. The mechanisms of participation of this complex matrix process in carcinogenesis are only starting to be appreciated. Recent studies showed key roles for stellate cells in the production of ECM proteins as well as cytokines and growth factors that promote the growth of the cancer cells all present in the desmoplastic parts of PDAC. In addition, interactions of ECM proteins and desmoplastic secreted growth factors with the cancer cells of PDAC activate intracellular signals including reactive oxygen species that act to make the cancer cells resistant to dying. These findings suggest that the desmoplasia of PDAC is a key factor in regulating carcinogenesis of PDAC as well as responses to therapies. A better understanding of the biology of desmoplasia in the mechanism of PDAC will likely provide significant opportunities for better treatments for this devastating cancer.

Neurotensin signaling induces intracellular alkalinization and interleukin-8 expression in human pancreatic cancer cells

Pancreatic adenocarcinomas express neurotensin receptors in up to 90% of cases, however, their role in tumor biology and as a drug target is not clear. In the present study, a stable neurotensin (NT) analog induced intracellular calcium release and intracellular alkalinization in BxPC-3 and PANC-1 pancreatic cancer cells that was abolished by inhibitors of NT receptor (NTR) and sodium–proton exchanger 1 (NHE1), amiloride and SR 142948, respectively. Activation of NHE1 involved increased phosphorylation of dimethylfumarate-sensitive mitogen- and stress-activated kinase 1/2 (MSK1/2). NTR signaling appears to promote a metastatic phenotype in pancreatic cancer cells by induction of localized extracellular acidification in normoxic cells, preceeding acidosis induced by hypoxia and switch to glycolysis in addition to increased expression of interleukin-8 (IL-8).

Blockade of SDF-1/CXCR4 signalling inhibits pancreatic cancer progression in vitro via inactivation of canonical Wnt pathway.

Extra-pancreatic metastasis is a difficult problem for surgical intervention in pancreatic cancer. CXC chemokine receptor 4 (CXCR4) was considered to have an important role in this process. We hypothesized it may contribute to the pancreatic cancer progression through influencing canonical Wnt pathway. The purpose of this study was to examine the functional role of CXCR4 in the progression of pancreatic cancers and explore the possible mechanism. To this end, the relation between CXCR4 and clinical characteristics was analysed. shRNA against CXCR4 was applied to disrupt the SDF-1/CXCR4 signal transduction pathways in pancreatic cancer cell lines. Our results showed that overall survival in the case of patients positive for CXCR4 expression was significantly lower than that in the case of patients negative for CXCR4 expression. Notably, in vitro studies we observed that the abrogation of CXCR4 could obviously influence the pancreatic cancer cell phenotype including cell proliferation, colony formation, cell invasion and also inhibit the TOPflash activity. In addition, Wnt target genes and mesenchymal markers such as Vimentin and Slug were also inhibited in CXCR4 knockdown cells. Collectively, these data reported here demonstrate CXCR4 could modulate the canonical Wnt pathway and perhaps be a promising therapeutic target for pancreatic cancer progression.

Divalent cations modulate the integrin‐mediated malignant phenotype in pancreatic cancer cells

We have previously demonstrated that pathophysiological shifts in the concentrations of extracellular Mg(2+) and Ca(2+) activate the alpha(2)beta(1) integrin-mediated malignant phenotype on type I collagen in pancreatic cancer cells, as evidenced by increased adhesion, migration and proliferation. In the present study, we examined the integrin and divalent cation specificity of pancreatic cancer cell interactions with other physiologically relevant extracellular matrix proteins, including fibronectin, type IV collagen, laminin and vitronectin. Our results indicate that, like alpha(2)beta(1) integrin-mediated interactions with type I collagen, beta(1) integrin-mediated adhesion to fibronectin, type IV collagen and laminin are promoted by Mg(2+) but not by Ca(2+). On vitronectin, cells attach via alpha(v)beta(5) and beta(1) integrins, and in the presence of either divalent cation. We also demonstrate that, like type I collagen, pancreatic cancer cell migration and proliferation on fibronectin, laminin and type IV collagen is maximal when Mg(2+) is present at concentrations that promote optimal adhesion and Ca(2+) is present at concentrations less than Mg(2+). On vitronectin, Panc-1 cell migration is maximal with decreased Mg(2+) and increased Ca(2+), but the reverse is true for BxPC-3 cells. Both cell lines exhibited maximal proliferation with increased Mg(2+) and decreased Ca(2+), however. Together with evidence indicating that the in vivo local tumor microenvironment contains increased Mg(2+) and decreased Ca(2+), our studies demonstrate that such divalent cation shifts could activate the integrin-mediated malignant phenotype in pancreatic cancer.

Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.

The RON Receptor Tyrosine Kinase Mediates Oncogenic Phenotypes in Pancreatic Cancer Cells and Is Increasingly Expressed during Pancreatic Cancer Progression

Pancreatic cancer is an aggressive disease characterized by rapid growth and early metastasis. The recepteur d'origine nantais (RON) receptor tyrosine kinase is overexpressed and/or constitutively active in several epithelial cancers, but its role in pancreatic cancer is unknown. In this study, we have characterized RON expression in both murine and human pancreatic cancer. Immunoblotting indicates that RON is expressed in pancreatic intraepithelial neoplasia (PanIN), primary, and metastatic cell lines both in the human and mouse. Immunostaining revealed that 93% of high-grade PanIN, 79% of primary, and 83% of metastatic lesions from human pancreatic tissue samples expressed RON, with minimal expression in normal ducts and low-grade PanIN (6% and 18%, respectively). Moreover, we show a dose-dependent effect of hepatocyte growth factor-like protein (HGFL), the RON-specific ligand, on pancreatic cancer cell migration and invasion, which was reversed by RON inhibition. Although stimulation with HGFL had no effect on proliferation, concurrent RON receptor blockade and gemcitabine treatment increased apoptosis of RON-expressing pancreatic cancer cells versus gemcitabine treatment alone. Finally, HGFL stimulation of pancreatic cancer cells resulted in increased expression of phospho-mitogen-activated protein kinase and phospho-Akt. Taken together, these findings suggest that RON receptor signaling may contribute to pancreatic carcinogenesis, and that further investigation is warranted to assess the potential of RON-directed therapies in this deadly disease.

Type I Collagen Promotes the Malignant Phenotype of Pancreatic Ductal Adenocarcinoma

The purpose of this study was to determine the role of functional interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) in the formation of the desmoplastic reaction (DR) in pancreatic cancer and to characterize the effect of type I collagen (the predominant component of the DR) on pancreatic cancer cell phenotype. PSCs and type I collagen were identified in sections of pancreatic cancer using immunohistochemistry, and their anatomic relationship was studied. Interactions among pancreatic cancer cell lines (MIA PaCa-2, Panc-1, and AsPC-1), primary cultures of human PSCs, and type I collagen were investigated in a series of tissue culture models. In vivo, the DR causes gross distortion of normal pancreas, bringing cancer cells into close contact with numerous PSCs and abundant type I collagen. In tissue culture models of pancreatic cancer, conditioned media from each cell line increased PSC [3H]thymidine incorporation up to 6.3-fold that of controls, and AsPC-1 cells also increased PSC collagen synthesis 1.3-fold. Type I collagen was observed to increase long-term survival of pancreatic cancer cells treated with 5-fluorouracil, by up to 62% in clonogenic assays. This was because type I collagen increased the proliferation of cancer cells ([3H]thymidine incorporation was up to 2.8-fold that of cells cultured on tissue culture plastic) and reduced apoptosis of AsPC-1 cells in response to 5-fluorouracil (by regulating mcl-1). These experiments elucidate a mechanism by which the DR in pancreatic cancer may form and, via the collagen within it, promote the malignant phenotype of pancreatic cancer cells, suggesting significant detriment to the host.

Treatment of pancreatic cancer cells with dicumarol induces cytotoxicity and oxidative stress.

quinone oxidoreductase (NQO(1)) catalyzes the two-electron reduction of quinones to hydroquinones. This reaction is believed to prevent the one-electron reduction of quinones that would result in redox cycling with generation of superoxide (O(2)(.-)). We have recently demonstrated that inhibition of NQO(1) with dicumarol increases intracellular O(2)(.-) production and inhibits the in vitro malignant phenotype of pancreatic cancer cells (J. Cullen et al., Cancer Res., 63: 5513-5520, 2003). We hypothesized that inhibition of NQO(1) would increase cell killing, induce oxidative stress, and inhibit in vivo tumor growth. In the human pancreatic cancer cell line MIA PaCa-2, dicumarol decreased cell viability, as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and decreased clonogenic survival. Dicumarol increased the percentage of apoptotic cells in a time-dependent and dose-dependent manner as measured by 3,3'-diaminobenzidine staining and flow cytometry, which was associated with cytochrome c release and poly(ADP-ribose) polymerase cleavage. Dicumarol also induced oxidative stress as evidenced by increased total glutathione and oxidized glutathione, as well as sensitizing to cell killing mediated by menadione. In established orthotopic pancreatic tumors in nude mice, intratumoral injections of dicumarol slowed tumor growth and extended survival. Inhibition of NQO(1) with dicumarol induces cell killing and oxidative stress in pancreatic cancer cells and speculate that dicumarol may prove to be useful in pancreatic cancer therapeutics.

Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions.

Radiotherapy represents a major treatment option for patients with pancreatic cancer, but recent evidence suggests that radiation can promote invasion and metastasis of cancer cells. Interactions between cancer cells and surrounding stromal cells may play an important role in aggressive tumor progression. In the present study, we investigated the invasive phenotype of pancreatic cancer cells in response to coculture with irradiated fibroblasts. Using in vitro invasion assay, we demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of pancreatic cancer cells and, surprisingly, the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. The hepatocyte growth factor (HGF) secretion from fibroblasts remained unchanged after irradiation, whereas exposure of pancreatic cancer cells to supernatant from irradiated fibroblasts resulted in increased phosphorylation of c-Met (HGF receptor) and mitogen-activated protein kinase activity, possibly or partially via increased expression of c-Met. We also demonstrated that scattering of pancreatic cancer cells was accelerated by the supernatant from irradiated fibroblasts. The enhanced invasiveness of pancreatic cancer cells induced by coculture with irradiated fibroblasts was completely blocked by NK4, a specific antagonist of HGF. These data suggest that invasive potential of certain pancreatic cancer cells is enhanced by soluble mediator(s) released from irradiated fibroblasts possibly through up-regulation of c-Met expression/phosphorylation and mitogen-activated protein kinase activity in pancreatic cancer cells. Our present findings further support the potential use of NK4 during radiotherapy for patients with pancreatic cancer.

Suppression of the Malignant phenotype in pancreatic cancer cells by the overexpression of glutathione peroxidase (GPx)

Suppression of the Malignant phenotype in pancreatic cancer cells by the overexpression of glutathione peroxidase (GPx)