CEACAM6 is a determinant of pancreatic adenocarcinoma cellular invasiveness

Pancreatic adenocarcinoma is among the most aggressively invasive malignancies. The immunoglobulin superfamily member carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers. We sought to define the role of CEACAM6 in pancreatic adenocarcinoma cellular invasiveness. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express low levels of CEACAM6. Retrovirally mediated RNA interference was used to silence CEACAM6 expression in BxPC3 cells, which inherently overexpress CEACAM6. Cellular invasiveness was quantified using a modified Boyden chamber assay. Overexpression of CEACAM6 increased Capan2 cellular invasiveness, whereas CEACAM6 knockdown attenuated BxPC3 invasiveness. A role for the c-Src tyrosine kinase in mediating CEACAM6-dependent invasiveness was defined using constitutively active and dominant-negative c-Src expression constructs. c-Src-dependent modulation of matrix metalloproteinase-9 activity contributes significantly to the increased cellular invasiveness induced by CEACAM6 overexpression. Levels of CEACAM6 expression can modulate pancreatic adenocarcinoma cellular invasiveness in a c-Src-dependent manner. This pathway warrants further investigation as a target for therapy.     

RNA interference demonstrates a novel role for integrin-linked kinase as a determinant of pancreatic adenocarcinoma cell gemcitabine chemoresistance.

Integrin-linked kinase (ILK) facilitates signal transduction between extracellular events and important intracellular survival pathways involving protein kinase B/Akt. We examined the role of ILK in determining pancreatic adenocarcinoma cellular chemoresistance to the nucleoside analogue gemcitabine. Cellular ILK expression was quantified by Western blot analysis. We examined the effects of overexpression of active ILK and of ILK knockdown induced by RNA interference on gemcitabine chemoresistance. We also examined the effects of modulating ILK expression on gemcitabine-induced caspase 3-mediated apoptosis, phosphorylation status of Akt (Ser473) and glycogen synthase kinase. Overexpression of ILK increased cellular gemcitabine chemoresistance, whereas ILK knockdown induced chemosensitization via increased caspase 3-mediated apoptosis. ILK knockdown attenuated Akt Ser473 and glycogen synthase kinase phosphorylation, whereas overexpression of constitutively active myristoylated Akt was sufficient to induce significant recovery in gemcitabine chemoresistance in the presence of ILK knockdown. Levels of ILK expression affect gemcitabine chemoresistance in pancreatic adenocarcinoma cells. This novel finding suggests that therapies directed against ILK and its downstream signaling targets may have the potential to enhance the efficacy of gemcitabine-based chemotherapy.     

Gemcitabine‐induced epithelial‐mesenchymal transition‐like changes sustain chemoresistance of pancreatic cancer cells of mesenchymal‐like phenotype

Growing body of evidence suggests that epithelial‐mesenchymal transition (EMT) is a critical process in tumor progression and chemoresistance in pancreatic cancer (PC). The aim of this study was to analyze the role of EMT‐like changes in acquisition of resistance to gemcitabine in pancreatic cells of the mesenchymal or epithelial phenotype. Therefore, chemoresistant BxPC‐3, Capan‐2, Panc‐1, and MiaPaca‐2 cells were selected by chronic exposure to increasing concentrations of gemcitabine. We show that gemcitabine‐resistant Panc‐1 and MiaPaca‐2 cells of mesenchymal‐like phenotype undergo further EMT‐like molecular changes mediated by ERK‐ZEB‐1 pathway, and that inhibition of ERK1/2 phosphorylation or ZEB‐1 expression resulted in a decrease in chemoresistance. Conversely, gemcitabine‐resistant BxPC‐3 and Capan‐2 cells of epithelial‐like phenotype did not show such typical EMT‐like molecular changes although the expression of the tight junction marker occludin could be found decreased. In pancreatic cancer patients, high ZEB‐1 expression was associated with tumor invasion and tumor budding. In addition, tumor budding was essentially observed in patients treated with neoadjuvant chemotherapy. These findings support the notion that gemcitabine treatment induces EMT‐like changes that sustain invasion and chemoresistance in PC cells.     

Overexpression of CEACAM6 promotes insulin-like growth factor I-induced pancreatic adenocarcinoma cellular invasiveness

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycosylphosphatidylinositol-linked immunoglobulin superfamily member that is overexpressed in a variety of human cancers. We have recently reported that suppression of CEACAM6 expression impairs pancreatic adenocarcinoma progression in vivo. In order to characterize the mechanisms through which CEACAM6 influences the malignant phenotype, CEACAM6-overexpressing Capan2 pancreatic adenocarcinoma cells were established by stable transfection. We determined the effect of CEACAM6 overexpression on cellular invasiveness towards insulin-like growth factor I (IGF-I), a peptide of critical importance in pancreatic cancer malignant cellular behavior and tumor progression. IGF-I-induced cellular invasiveness and IGF-IR expression were significantly increased in clones overexpressing CEACAM6. Using inhibitory anti-IGF-IR antibody, a requirement for IGF-IR signaling in the enhanced invasiveness towards IGF-I induced by CEACAM6 overexpression was confirmed. CEACAM6-overexpressing clones exhibited increased Akt and c-Src kinase activities, as well as higher levels of matrix metalloproteinase-2 (MMP-2) expression and activity in the presence of IGF-I. While Akt kinase is both necessary and sufficient to induce IGF-IR upregulation, c-Src kinase activity is necessary, but alone is insufficient to upregulate IGF-IR expression. CEACAM6 is an important determinant of pancreatic adenocarcinoma malignant cellular behavior and, together with its downstream targets, warrants further investigation as a therapeutic target in this disease.     

siRNA directed against TrkA sensitizes human pancreatic cancer cells to apoptosis induced by gemcitabine through an inactivation of PI3K/Akt-dependent pathway

Previously, we have documented that the aggressive and highly metastatic behavior of pancreatic cancer may be due to the aberrant expression of nerve growth factor (NGF) and its high-affinity receptor, proto-oncogene TrkA. In this study, we sought to determine the effect of suppressing TrkA expression on pancreatic cancer chemosensitivity to gemcitabine. Human pancreatic cancer cell lines PANC-1, MIA-PaCa-2 and ASPC-1 were studied. The expression and kinase activity of TrkA were determined by Western blot analysis and in vitro kinase assay respectively. RNA interference was used to suppress TrkA expression. Gemcitabine-induced cytotoxicity was determined by tetrazolium reduction assay and caspase profiling was performed. The effect of TrkA-specific siRNA on PI3K/Akt activity was also quantified. TrkA expression and kinase activity in cell lines were directly correlated with gemcitabine chemoresistance. TrkA-specific siRNA suppressed TrkA expression and kinase activity, and furthermore increased gemcitabine-induced, caspase-mediated apoptosis. PI3K/Akt activity was decreased by suppression of TrkA expression. Taken together, these data demonstrated that TrkA is a determinant of pancreatic adenocarcinoma chemoresistance and PI3K/Akt is a key signaling component by which NGF activation of the TrkA signal transduction pathway protects pancreatic cancer cells from chemotherapy-induced cell death.     

CEACAM6 gene silencing impairs anoikis resistance and in vivo metastatic ability of pancreatic adenocarcinoma cells

Anoikis is the apoptotic response induced in normal cells by inadequate or inappropriate adhesion to substrate. It is postulated that resistance to anoikis facilitates tumorigenesis and metastasis. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member overexpressed in a number of human cancers and implicated in anoikis resistance. We tested the effect of CEACAM6 gene silencing on anoikis in pancreatic adenocarcinoma cell lines. Anoikis was induced in PANC1, Capan2, MiaPaCa2 and Mia(AR) (a MiaPaCa2-derived anoikis-resistant subline) by culture in poly-2-hydroxyethylmethacrylate-coated wells. Anoikis was quantified by YO-PRO-1/propidium iodide staining and flow cytometry. The role of caspase activation was determined using fluorometric profiling and the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk). CEACAM6 expression was suppressed by RNA interference. Using a nude mouse orthotopic xenograft model, we assessed the effect of this treatment on in vivo metastatic ability. Anoikis resistance was associated with increased CEACAM6 expression. CEACAM6-specific short interfering ribonucleic acid (siRNA), but not control siRNA, increased susceptibility to caspase-mediated anoikis, an effect abrogated by Z-VAD-fmk, and decreased Akt phosphorylation (Ser-473) under anchorage-independent conditions. CEACAM6 gene silencing reversed the acquired anoikis resistance of Mia(AR) and inhibited its in vivo metastatic ability. CEACAM6 warrants further investigation as a novel therapeutic target for the treatment of pancreatic adenocarcinoma.     

Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation,migration, invasion and angiogenesis of pancreatic cancer cells

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is over-expressed in pancreatic cancer cells, and it is associated with the progression of pancreatic cancer. We tested a single domain antibody (sdAb) targeting CEACAM6, 2A3, which was isolated previously from a llama immune library, and an Fc conjugated version of this sdAb, to determine how they affect the pancreatic cancer cell line BxPC3. We also compared the effects of the antibodies to gemcitabine. Gemcitabine and 2A3 slowed down cancer cell proliferation. However, only 2A3 retarded cancer cell invasion, angiogenesis within the cancer mass and BxPC3 cell MMP-9 activity, three features important for tumour growth and metastasis. The IC₅₀s for 2A3, 2A3-Fc and gemcitabine were determined as 6.5 μM, 8 μM and 12 nM, respectively. While the 2A3 antibody inhibited MMP-9 activity by 33% compared to non-treated control cells, gemcitabine failed to inhibit MMP-9 activity. Moreover, 2A3 and 2A3-Fc inhibited invasion of BxPC3 by 73% compared to non-treated cells. When conditioned media that were produced using 2A3- or 2A3-Fc-treated BxPC3 cells were used in a capillary formation assay, the capillary length was reduced by 21% and 49%, respectively. Therefore 2A3 is an ideal candidate for treating tumours that over-express CEACAM6.     

RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine

Ribonucleotide reductase is emerging as an important determinant of gemcitabine chemoresistance in human cancers. Activity of this enzyme, which catalyses conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotides, is modulated by levels of its M2 subunit (RRM2). Here we show that RRM2 overexpression is associated with gemcitabine chemoresistance in pancreatic adenocarcinoma cells, and that suppression of RRM2 expression using RNA interference mediated by small interfering RNA (siRNA) enhances gemcitabine-induced cytotoxicity in vitro. We demonstrate the ability of systemically administered RRM2 siRNA to suppress tumoral RRM2 expression in an orthotopic xenograft model of pancreatic adenocarcinoma. Synergism between RRM2 siRNA and gemcitabine results in markedly suppressed tumor growth, increased tumor apoptosis and inhibition of metastasis. Our findings confirm the importance of RRM2 in pancreatic adenocarcinoma gemcitabine chemoresistance. This is the first demonstration that systemic delivery of siRNA-based therapy can enhance the efficacy of an anticancer nucleoside analog.     

miR-203 reverses chemoresistance in p53-mutated colon cancer cells through downregulation of Akt2 expression

In this study, we explored miR-203’s role in the chemoresistance of colon cancer. We found that overexpression of miR-203 significantly decreased cell proliferation and survival, and induced cell apoptosis in the p53-mutated colon cancer cells. Importantly, miR-203 overexpression increased the cytotoxic role of paclitaxel in the p53-mutated colon cancer cells, but not in the p53 wild-type cells. We further demonstrated that the tumor suppressive role of miR-203 was mediated by negatively regulating Akt2 protein expression through mRNA degradation. The inhibition of Akt2 activity downregulated the protein expression of its downstream molecules involved in chemoresistance, such as MTDH and HSP90 genes. Also, overexpression of miR-203 decreased anti-apoptotic gene Bcl-xL expression and increased apoptotic proteins Bax and active caspase-3 levels. Our study is the first to identify the tumor suppressive role of overexpressed miR-203, describe its associated signaling pathways, and highlight the role of miR-203 in chemoresistance.     

Arsenic trioxide-induced apoptosis in U937 cells involve generation of reactive oxygen species and inhibition of Akt

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in acute promyelocytic leukemia (APL), but little is known about the molecular mechanisms mediating these effects. In the present study, we determined the molecular pathways that lead to apoptosis after treatment of cells with arsenic trioxide. Arsenic trioxide treatment of U937 cells leads to apoptosis, which is accompanied by activation of caspase 3 (as measured by decreased levels of the 32 kDa inactive form and increased proteolytic cleavage of PLC-gamma1). The broad-range caspase inhibitor z-VAD-fmk inhibits this induction of apoptosis, supporting a direct link between caspase activation and arsenic trioxide induction of apoptosis. This activation of apoptosis is accompanied by release of cytochrome c, down-regulation of cIAP1, and inactivation of Akt. Bcl-2 overexpression attenuates arsenic trioxide-induced apoptosis in U937 cells by inhibition of caspase 3 activity, but not inhibition of Akt. In addition, arsenic trioxide-induced apoptosis was caused by the generation of reactive oxygen species, which was prevented by antioxidant NAC (N-acetyl-cysteine). Co-treatment with NAC markedly prevented dephosphorylation of Akt, activation of caspase 3, and down-regulation of cIAP1. These data indicate that arsenic trioxide can cause cell damage by inactivating the Akt-related cell survival pathway and generating the reactive oxygen species, providing a new mechanism for arsenic trioxide-induced apoptosis.     

EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human cancers. We sought to characterize the role of EphA2 in pancreatic adenocarcinoma and, using RNA interference (RNAi) mediated by small interfering RNA (siRNA), we determined the effects of suppressing EphA2 expression in vitro and in vivo. EphA2 expression in PANC1, MIAPaCa2, BxPC3 and Capan2 cells was assessed by Northern and Western blot. We artificially overexpressed EphA2 by transient transfection and suppressed EphA2 expression using RNAi. Cellular invasiveness was quantified by modified Boyden chamber assay. Anoikis was induced by anchorage-independent polyHEMA culture and caspase 3 activity was quantified fluorometrically. Focal adhesion kinase (FAK) phosphorylation was assessed by immunoprecipitation. EphA2 siRNA treatment was assessed in a nude mouse xenograft model. Pancreatic adenocarcinoma cells differentially express EphA2. Inherent and induced EphA2 overexpression is associated with increased cellular invasiveness and anoikis resistance. EphA2 siRNA suppresses EphA2 expression, cellular invasiveness, anoikis resistance and FAK phosphorylation in vitro and retards tumor growth and inhibits metastasis in vivo. EphA2 is both a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma.     

Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species,down-regulation of Bcl-XL and IAP,the release of cytochrome c and inhibition of Akt

Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. The mechanism by which curcumin initiates apoptosis remains poorly understood. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human renal Caki cells. Treatment of Caki cells with 50 microM curcumin resulted in the activation of caspase 3, cleavage of phospholipase C-gamma1 and DNA fragmentation. Curcumin-induced apoptosis is mediated through the activation of caspase, which is specifically inhibited by the caspase inhibitor, benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone. Curcumin causes dose-dependent apoptosis and DNA fragmentation of Caki cells, which is preceded by the sequential dephosphorylation of Akt, down-regulation of the anti-apoptotic Bcl-2, Bcl-XL and IAP proteins, release of cytochrome c and activation of caspase 3. Cyclosporin A, as well as caspase inhibitor, specifically inhibit curcumin-induced apoptosis in Caki cells. Pre-treatment with N-acetyl-cysteine, markedly prevented dephosphorylation of Akt, and cytochrome c release, and cell death, suggesting a role for reactive oxygen species in this process. The data indicate that curcumin can cause cell damage by inactivating the Akt-related cell survival pathway and release of cytochrome c, providing a new mechanism for curcumin-induced cytotoxicity.     

XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells

Chemoresistance is a major hurdle for successful cancer therapy. Although multiple mechanisms have been implicated to be involved in cisplatin resistance, recent evidence has suggested that X-linked inhibitor of apoptosis protein (XIAP) may be a key determinant in chemosensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and apoptotic signaling. Whereas phosphatidylinositol 3-kinase (PI 3-K) and XIAP are believed to be important cell survival factors in human ovarian surface epithelial cancer cells, if and how they interact to confer resistance to chemotherapy is not known. In the present study, we have investigated the role of XIAP in the regulation of the PI 3-K/Akt survival pathway in chemosensitive (A2780-s, OV2008, and OVCAR-3) and resistant (A2780-cp) ovarian cancer cell lines and the nature of this interaction in cell death/survival signaling. Cisplatin decreased XIAP protein levels and induced Akt cleavage and apoptosis in chemosensitive, but not in resistant, ovarian cancer cells. Cisplatin also induced cleavage of caspase-9 and caspase-3, a process blocked by XIAP overexpression. Pretreatment of ovarian cancer cells and their whole cell lysate with tetrapeptide inhibitors of caspases in vitro significantly decreased Akt cleavage induced by cisplatin and exogenous active caspase-3. Adenoviral sense XIAP cDNA expression increased XIAP protein levels and increased Akt phosphorylation, indicative of activation of Akt and, likely, of PI 3-K. This was associated with a decrease in cisplatin-induced apoptosis. In a cell line (OVCAR-3) where basal phosphorylated Akt levels were high, XIAP overexpression failed to increase further the level of this phosphoprotein. XIAP down-regulation induced Akt cleavage and apoptosis, and treatment of whole cell lysate with human recombinant active caspase-3 resulted in a similar pattern of Akt cleavage. In the presence of the PI 3-K inhibitor (LY294002), XIAP overexpression failed to block cisplatin-induced apoptosis and to induce Akt phosphorylation, suggesting that the site of action of XIAP is upstream of Akt in this cell survival pathway. Taken together, the results indicate that XIAP prevents apoptosis through a PI 3-K-dependent inhibition of the caspase cascade. These results demonstrate a novel mechanism by which XIAP regulates apoptosis and the possible involvement of the PI 3-K/Akt survival pathway in XIAP-mediated chemoresistance of ovarian cancer cells.     

Growth factor-independent activation of protein kinase B contributes to the inherent resistance of vascular endothelium to radiation-induced apoptotic response

The phosphatidylinositol 3'-kinase (PI3K)/protein kinase B (Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival during oxidative stress. The role of the PI3K/Akt pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was phosphorylated rapidly in response to radiation in primary culture human umbilical vein endothelial cells in the absence of serum or growth factors. Akt phosphorylation occurred after doses as low as 1 Gy. PI3K consists of p85 and p110 subunits, which play a central role in Akt activation in response to exogenous stimuli. A mutation within the Src homology region 2 domain of mutant p85 (Deltap85) prevented radiation-induced Akt phosphorylation, when overexpressed in endothelial cells. Vascular endothelial cells transduced with control vector were resistant to radiation-induced apoptosis, whereas endothelial cell transduction with adenovirus encoding the mutated p85 (Ad.Deltap85) reversed this resistance to apoptosis after treatment with intermediate radiation doses (2-6 Gy). Deltap85 overexpression alone had no effect on the viability or apoptosis of endothelial cells. However, irradiated endothelial cells overexpressing Deltap85 released cytochrome c into the cytosol fraction and activated proteolytic cleavage of caspases 3 and 9, thereby inducing the apoptotic response. Inhibition of caspase 3 blocked endothelial apoptosis induced by overexpression of Deltap85 and radiation. These findings suggest that growth factor-independent activation of Akt contributes, in part, to the inherent resistance of irradiated vascular endothelium to the activation of apoptotic response.     

CEACAM6 gene expression in intrahepatic cholangiocarcinoma

The purpose of this study was to investigate the clinicopathological and biological significance of human carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) gene expression in human intrahepatic cholangiocarcinoma. CEACAM6 is reported to be involved in human malignancies. However, in cholangiocarcinoma expression of CEACAM6 and its clinicopathological significance have not been investigated. CEACAM6 expression status was determined and analysed with respect to various clinicopathological parameters in 23 intrahepatic cholangiocarcinomas. Additionally, we investigated effects of CEACAM6 gene in the cholangiocarcinoma cell lines. CEACAM6 gene expression in cancer tissues was higher than in noncancerous tissues in 16 of the 23 cases; however, it was not statistically significant. The tumours with elevated CEACAM6 expression showed a tendency to be associated with lymphatic invasion and stage of the disease. Interestingly, patients with high CEACAM6 expression showed a significantly poorer disease-free survival rate than those with low CEACAM6 expression. We demonstrated that CEACAM6-transfected cells were more proliferative, more invasive and more chemoresistant to gemcitabine compared to mock-transfected cells. Furthermore, CEACAM6 gene silencing by CEACAM6-specific siRNA resulted in higher chemosensitivity to gemcitabine. CEACAM6 is a potential prognostic indicator and potential chemoresistant marker to gemcitabine for patients with intrahepatic cholangiocarcinoma.     

Inhibition of Notch3 enhances sensitivity to gemcitabine in pancreatic cancer through an inactivation of PI3K/Akt-dependent pathway

Notch3 is one of the four Notch receptors identified in mammal, but its role in human pancreatic cancer remains poorly characterized. In this study, we sought to determine the effect of suppressing Notch3 expression on the chemosensitivity to gemcitabine in human pancreatic cancer cell lines BxPC-3 and PANC-1. RNA interference was used to suppress Notch3 expression. Gemcitabine-induced cytotoxicity was determined by MTT. Cell apoptosis was measured by flow cytometry. Caspase 3 activity was assayed using a Caspase Fluorescent Assay Kit. The effect of Notch3-specific siRNA on PI3K/Akt activity was also quantified. Notch3-specific siRNA suppressed Notch3 expression, and furthermore increased gemcitabine-induced, caspase-mediated apoptosis. The suppression of Notch3 expression decreased the average IC₅₀ in BxPC-3 and PANC-1 cells treated with gemcitabine. PI3K/Akt activity was decreased by the suppression of Notch3 expression. Taken together, these data demonstrated that Notch3 is a potential therapeutic target for pancreatic cancer, and PI3K/Akt is a key signaling component by which activation of the Notch3 signal transduction pathway protects pancreatic cancer cells from chemotherapy-induced cell death.