Homo sapiens systemic RNA interference-defective-1 transmembrane family member 1 (SIDT1) protein mediates contact-dependent small RNA transfer and microRNA-21-driven chemoresistance.

Locally initiated RNA interference (RNAi) has the potential for spatial propagation, inducing posttranscriptional gene silencing in distant cells. In Caenorhabditis elegans, systemic RNAi requires a phylogenetically conserved transmembrane channel, SID-1. Here, we show that a human SID-1 orthologue, SIDT1, facilitates rapid, contact-dependent, bidirectional small RNA transfer between human cells, resulting in target-specific non-cell-autonomous RNAi. Intercellular small RNA transfer can be both homotypic and heterotypic. We show SIDT1-mediated intercellular transfer of microRNA-21 to be a driver of resistance to the nucleoside analog gemcitabine in human adenocarcinoma cells. Documentation of a SIDT1-dependent small RNA transfer mechanism and the associated phenotypic effects on chemoresistance in human cancer cells raises the possibility that conserved systemic RNAi pathways contribute to the acquisition of drug resistance. Mediators of non-cell-autonomous RNAi may be tractable targets for novel therapies aimed at improving the efficacy of current cytotoxic agents.

Giant haemangioma of the liver: observation or resection?

BACKGROUND: Haemangiomata are the most frequent benign solid liver lesion. The management of giant (> or =5 cm) haemangiomata of the liver remains controversial. METHODS: A search of relevant peer-reviewed literature was conducted using PubMed and original articles were reviewed. RESULTS AND CONCLUSIONS: The vast majority of giant haemangiomata remain asymptomatic and have a benign and uncomplicated natural history. Decisions regarding the optimal management of giant haemangiomata depend on a high level of confidence in diagnostic imaging. Diagnostic biopsy to differentiate giant haemangiomata from malignant lesions should be discouraged. Despite limitations and alternative modalities, surgery remains the only consistently effective curative treatment for giant haemangiomata. Surgery is not generally justified to prevent complications in asymptomatic patients. Principal indications for the surgical management of giant haemangiomata include established complications, incapacitating symptoms and uncertainty of diagnosis. Patients should only be selected for surgery based on a careful assessment of risks and benefits of intervention.

RRM2 induces NF-kappaB-dependent MMP-9 activation and enhances cellular invasiveness.

Ribonucleotide reductase is a dimeric enzyme that catalyzes conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotide forms, a rate-limiting step in the production of 2'-deoxyribonucleoside 5'-triphosphates required for DNA synthesis. The ribonucleotide reductase M2 subunit (RRM2) is a determinant of malignant cellular behavior in a range of human cancers. We examined the effect of RRM2 overexpression on pancreatic adenocarcinoma cellular invasiveness and nuclear factor-kappaB (NF-kappaB) transcription factor activity. RRM2 overexpression increases pancreatic adenocarcinoma cellular invasiveness and MMP-9 expression in a NF-kappaB-dependent manner. RNA interference (RNAi)-mediated silencing of RRM2 expression attenuates cellular invasiveness and NF-kappaB activity. NF-kappaB is a key mediator of the invasive phenotypic changes induced by RRM2 overexpression.

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.

RNA interference: a mammalian SID-1 homologue enhances siRNA uptake and gene silencing efficacy in human cells.

SID-1 is a transmembrane protein that mediates systemic RNA interference in Caenorhabditis elegans. Here we show that the mammalian SID-1 homologue FLJ20174 localizes to the cell membrane of human cells and enhances their uptake of small interfering RNA (siRNA), resulting in increased siRNA-mediated gene silencing efficacy. This is the first demonstration to show that overexpression of a membrane protein enhances siRNA internalization in mammalian cells. This observation raises the possibility of enhancing the efficacy of RNA interference.

CEACAM6 is a novel biomarker in pancreatic adenocarcinoma and PanIN lesions.

OBJECTIVE: The purpose of this study was to test the hypothesis that CEACAM6 expression is an indicator of adverse pathologic features and clinical outcome in pancreatic adenocarcinoma. SUMMARY BACKGROUND DATA: Previously, we have demonstrated carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) to be an oncoprotein that plays an important role in the biology of pancreatic adenocarcinoma. Suppression of CEACAM6 expression reduces tumorigenesis and metastasis in vivo. METHODS: A tissue microarray was constructed using tumor specimens obtained from 89 consecutive patients who had undergone pancreatic resection for pancreatic adenocarcinoma with curative intent. A second microarray containing 54 pancreatic intraepithelial neoplasia (PanIN) lesions was constructed using tissues from a separate cohort of 44 patients. Both arrays were immunostained using a specific anti-CEACAM6 monoclonal antibody. Tumoral CEACAM6 expression was dichotomized into negative and positive immunoreactivity groups. The log-rank test was used to evaluate univariate associations of CEACAM6 expression with prognosis. Survival curves were derived using the Kaplan-Meier method. RESULTS: Tumoral CEACAM6 expression was detected in 82 (92%) pancreatic adenocarcinoma specimens. CEACAM6 expression was more prevalent in high-grade than in low-grade PanIN lesions (P = 0.0002). Negative tumoral CEACAM6 expression was associated with absence of lymph node metastases (P = 0.012), lower disease stage (P = 0.008), and longer postoperative survival (P = 0.047). CONCLUSIONS: CEACAM6 is a novel biomarker for pancreatic adenocarcinoma. CEACAM6 warrants further evaluation as both a prognostic factor and a therapeutic target in pancreatic cancer.

Systemic siRNA-mediated gene silencing: a new approach to targeted therapy of cancer.

OBJECTIVE: RNA interference (RNAi), mediated by small interfering RNA (siRNA), silences genes with a high degree of specificity and potentially represents a general approach for molecularly targeted anticancer therapy. The aim of this study was to evaluate the ability of systemically administered siRNA to silence gene expression in vivo and to assess the effect of this approach on tumor growth using a murine pancreatic adenocarcinoma xenograft model. SUMMARY BACKGROUND DATA: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is widely overexpressed in human gastrointestinal cancer. Overexpression of CEACAM6 promotes cell survival under anchorage independent conditions, a characteristic associated with tumorigenesis and metastasis. METHODS: CEACAM6 expression was quantified by real-time polymerase chain reaction (PCR) and Western blot. Mice (n = 10/group) were subcutaneously xenografted with 2 x 10 BxPC3 cells (which inherently overexpress CEACAM6). Tumor growth, CEACAM6 expression, cellular proliferation (Ki-67 immunohistochemistry), apoptosis, angiogenesis (CD34 immunohistochemistry), and survival were compared for mice administered either systemic CEACAM6-specific or control single-base mismatch siRNA over 6 weeks, following orthotopic tumor implantation. RESULTS: Treatment with CEACAM6-specific siRNA suppressed primary tumor growth by 68% versus control siRNA (P < 0.05) and was associated with a decreased proliferating cell index, impaired angiogenesis and increased apoptosis in the xenografted tumors. CEACAM6-specific siRNA completely inhibited metastasis (0% of mice versus 60%, P < 0.05) and significantly improved survival, without apparent toxicity. CONCLUSIONS: Our data demonstrate the efficacy of systemically administered siRNA as a therapeutic modality in experimental pancreatic cancer. This novel therapeutic strategy may be applicable to a broad range of cancers and warrants investigation in patients with refractory disease.

Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: A novel therapeutic strategy in pancreatic cancer.

BACKGROUND: Ribonucleotide reductase M2 subunit (RRM2) overexpression enhances tumor chemoresistance and cellular invasiveness. We hypothesized that the RNA interference (RNAi) induced by retrovirally delivered small interfering RNA (siRNA) would sensitize pancreatic adenocarcinoma cells to gemcitabine and attenuate their invasive potential. METHODS: Stable suppression of RRM2 expression in PANC1, MIAPaCa2, BxPC3, and Capan2 cells was induced by exposure to a novel replication-deficient retrovirus, engineered to express RRM2-specific siRNA (psiRRM2), and confirmed by Western blot analysis. Single-base mismatch vector (psiControl) served as control. Ribonucleotide reductase activity was quantified, and gemcitabine 50% inhibitory concentrations were calculated. TUNEL staining and caspase profiling were performed after gemcitabine exposure. Cellular invasiveness was quantified in a Matrigel Boyden chamber. NF-kappaB activity and matrix metalloproteinase-9 (MMP-9) expression and activity were measured. RESULTS: RRM2 expression was stably and specifically suppressed in psiRRM2, but not psiControl transfectants. psiRRM2 transfectants exhibited lower 50% inhibitory concentrations, increased gemcitabine-induced apoptosis, and greater caspase-3 activation, relative to psiControl transfectants. Invasiveness was attenuated in psiRRM2 transfectants, as was NF-kappaB activity, MMP-9 expression, and MMP-9 activity, relative to psiControl transfectants. CONCLUSIONS: RRM2 gene silencing attenuates pancreatic adenocarcinoma cellular invasiveness and gemcitabine chemoresistance. Retroviral siRNA delivery can efficiently induce stable RNAi, allowing dissection of gene function and potentially representing a new therapeutic modality.

Ligation of EphA2 by Ephrin A1-Fc inhibits pancreatic adenocarcinoma cellular invasiveness.

The Eph tyrosine kinases interact with ligands of the Ephrin family and have diverse cellular functions. EphA2 has been recognized to be an oncoprotein of importance in a range of cancers. Here, we examine the effect of EphA2 overexpression and ligation by chimeric Ephrin A1-Fc on the invasive phenotype of pancreatic adenocarcinoma cells. We show that EphA2 overexpression induces a FAK-dependent increase in MMP-2 expression and invasiveness. EphA2 ligation induces proteosomal degradation of EphA2, attenuates the invasive phenotype, and decreases both FAK phosphorylation and MMP-2 expression. EphA2 appears to represent a rational therapeutic target and ligation by Ephrin A1-Fc is one strategy to modulate levels of this oncoprotein.

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 c-Src enhances pancreatic adenocarcinoma cell gemcitabine chemosensitivity.

BACKGROUND: The c-Src tyrosine kinase is a determinant of malignant cellular behavior in a variety of human cancers. We sought to determine the effect of suppressing c-Src expression on pancreatic adenocarcinoma chemosensitivity to gemcitabine. STUDY DESIGN: PANC1, MIAPaCa2, BxPC3, and Capan2 pancreatic adenocarcinoma cell lines were studied. Expression of c-Src was determined by Western blot analysis. c-Src kinase activity was determined by in vitro kinase assay. RNA interference was used to suppress c-Src expression. Gemcitabine-induced cytotoxicity was determined by tetrazolium reduction assay and caspase profiling was performed. The effect of Src-specific siRNA on Akt activity was quantified. RESULTS: Src expression and kinase activity in cell lines were directly correlated with gemcitabine chemoresistance. c-Src-specific siRNA suppressed c-Src expression and kinase activity. c-Src-specific siRNA increased gemcitabine-induced, caspase-mediated apoptosis. Akt activity was decreased by suppression of c-Src expression. CONCLUSIONS: c-Src is a determinant of pancreatic adenocarcinoma chemoresistance and represents a potential target for therapeutic intervention.

A novel role for carcinoembryonic antigen-related cell adhesion molecule 6 as a determinant of gemcitabine chemoresistance in pancreatic adenocarcinoma cells.

Most patients with pancreatic adenocarcinoma present with surgically incurable disease. Gemcitabine, the principal agent used to treat such patients, has little impact on outcome. Overexpression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, a feature of this malignancy, is associated with resistance to anoikis and increased metastasis. The purpose of this study was to determine the role of CEACAM6 in cellular chemoresistance to gemcitabine. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express very low levels of the protein. Suppression of CEACAM6 expression was achieved in BxPC3 cells, which inherently overexpress CEACAM6, by stable transfection of a CEACAM6 small interfering RNA-generating vector. The effects of modulating CEACAM6 expression on gemcitabine-induced cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay, flow cytometric apoptosis quantification, caspase profiling, and Western analysis of cytoplasmic cytochrome c release. The roles of Akt and c-Src kinases as downstream targets of CEACAM6 signaling were examined. Stable overexpression of CEACAM6 in Capan2 increased gemcitabine chemoresistance, whereas CEACAM6 gene silencing in BxPC3 markedly increased the sensitivity of these cells to gemcitabine. Differential expression of CEACAM6 modulates Akt activity in a c-Src-dependent manner, and CEACAM6 overexpression appears to protect cells from cytochrome c-induced caspase 3 activation and apoptosis.

Inhibition of SRC tyrosine kinase impairs inherent and acquired gemcitabine resistance in human pancreatic adenocarcinoma cells.

PURPOSE: We tested the hypotheses that Src tyrosine kinase overactivity represents a chemoresistance mechanism and that Src inhibition may enhance gemcitabine cytotoxicity in pancreatic adenocarcinoma cells. EXPERIMENTAL DESIGN: Pancreatic adenocarcinoma cells PANC1, MiaPaCa2, Capan2, BxPC3, and PANC1(GemRes), a stably gemcitabine-resistant subline of PANC1, were exposed to combinations of gemcitabine and Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Src expression, phosphorylation (Tyr-416), and activity were analyzed by immunoblotting and in vitro kinase assay. Expression of the M2 subunit of ribonucleotide reductase (RRM2), a putative chemoresistance enzyme, was quantified by Northern and Western blot. Cellular proliferation was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was characterized by YO-PRO-1/propidium iodide staining, fluorometric caspase profiling, and caspase inhibition (Z-Val-Ala-Asp-fluoromethyl ketone). The effects of constitutively active and dominant negative Src were determined. The therapeutic efficacy of PP2 in combination with gemcitabine was tested in nude mice orthotopically xenografted with PANC1(GemRes). RESULTS: Greater gemcitabine resistance was associated with higher Src phosphorylation and activity, both of which were higher in PANC1(GemRes), relative to PANC1; total Src levels were alike. PANC1(GemRes) overexpressed RRM2. PP2 enhanced inherent gemcitabine chemosensitivity and attenuated gemcitabine resistance in PANC1(GemRes). Constitutively active Src increased gemcitabine chemoresistance; dominant negative Src impaired gemcitabine chemoresistance. PP2 augmented gemcitabine-induced caspase-mediated apoptosis, suppressed RRM2 expression, and decreased activity of the RRM2-regulating transcription factor E2F1 in PANC1(GemRes). PP2 and gemcitabine in combination substantially decreased tumor growth and inhibited metastasis in vivo. CONCLUSIONS: Increased Src tyrosine kinase activity represents a potential chemoresistance mechanism and a promising therapeutic target warranting further investigation in gemcitabine-resistant pancreatic adenocarcinoma.