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.     

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.     

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.     

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.     

Tumor growth inhibition by synthetic and expressed siRNA targeting focal adhesion kinase

Focal adhesion kinase (FAK) was first identified as a viral Src substrate, and substantial experimental data have significantly correlated the elevated FAK expression in human tumor cells with an increased cell adhesion and invasion potential. However, studies investigating the role of FAK in cell proliferation have been limited. Recently, a technique known as RNA interference (RNAi) was successfully adapted to mammalian cells to decrease specifically the expression of targeted cellular genes. In this study, we investigated the role of FAK in cell proliferation, adhesion, and migration by using small interfering RNA (siRNA) technique. Firstly, we constructed a plasmid library expressing short hairpin RNAs (shRNAs) targeting FAK and selected clones substantially suppressing FAK expression in HeLa and HT1080 cells. We then studied the function of FAK in the highly invasive human prostate cancer cell line, PC3M, and mouse breast cancer cell line 4T1, by using selected shRNA clones (#40 and #42) and siRNAs chemically synthesized following the target sequences of #40 and #42. We demonstrated that the decrease of FAK protein expression by treatment with shRNA/siRNA targeting FAK inhibited cell adhesion on a fibronectin/laminin-coated plate, cell migration in a haptotactic migration assay, and cell proliferation in vitro. Furthermore, it suppressed tumor growth in vivo in heterotopic/orthotopic mice models. These results support our hypothesis that FAK plays a crucial role in tumor formation and growth in vivo by regulation of cell adhesion and proliferation by FAK-dependent signals.     

Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer

Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.     

黏着斑激酶在骨肉瘤中的表达及临床意义

  目的   探讨骨肉瘤组织中黏着斑激酶（focal adhesion kinase,FAK）的表达情况及其与临床病理特征和预后的关系。  方法   收集1999年12月至2011年l2月东南大学附属中大医院和南京军区南京总医院收治的经手术病理确诊的骨肉瘤标本113例,免疫组织化学检测病灶中FAK表达量及磷酸化水平,并通过RNA干扰观察改变FAK的表达与激活水平对骨肉瘤细胞增殖、凋亡、迁移及侵袭活动的影响。  结果  70例标本（61.95%）FAK过表达,其中42例（37.17%）pFAK阳性。FAK表达谱与性别、年龄、AJCC ⅡA/ⅡB期、病灶部位、术式、术前化疗效果等临床病理参数均无显著相关性。生存分析显示FAK的过表达及其磷酸化显著缩短了总生存时间（overall survival,OS）和无转移生存时间（metastasis-free survival,mFS）,其与术前化疗效果是预测骨肉瘤生存时间和转移早晚的两个独立预后因素。细胞学实验则显示FAK的表达和激活可以促进骨肉瘤细胞的增殖、迁移及侵袭活动,并能抑制凋亡。  结论  FAK的过表达及其磷酸化与骨肉瘤恶性程度密切相关,可为生存期及预后判断提供一定参考。      

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.     

A small interfering RNA targeting osteopontin as gastric cancer therapeutics

It has been shown that Osteopontin (OPN) protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of OPN, RNA interference (RNAi) was used to inhibit OPN expression in the human gastric cancer cells in vitro and in vivo. BGC-823, gastric cancer cell line, was stably transfected with OPN small interfering RNA (siRNA) plasmids. OPN siRNA significantly reduced the expression of OPN in human gastric cancer cells in transient- and stable-transfection manner. In vitro down-regulation of OPN inhibited BGC-823 cell growth, anchorage-independent growth, migration and invasion. The results further showed that OPN small interfering RNA suppressed the growth, migration and invasion of gastric cancer cell through the reduction of MMP-2 and uPA expression, inhibition of NF-kappaB DNA binding activity, and down-regulation of Akt phosphorylation. In vivo animal studies showed that tumor growth was significantly inhibited in OPN siRNA group compared with WT. Intratumor gene therapy with polyethylenimine/OPNsi also resulted in tumor growth suppression and prolonged survival. Thus, this study demonstrated that down-regulation of OPN could suppress the growth, migration and invasion of gastric cancer cells, and OPN siRNA may offer a new potential gene therapy approach for human gastric cancer in future.     

Re-expression of the tumor suppressor NF2/merlin inhibits invasiveness in mesothelioma cells and negatively regulates FAK

The neurofibromatosis type 2 NF2 gene product, merlin, is a tumor suppressor frequently inactivated in malignant mesothelioma (MM). To investigate a possible correlation between merlin inactivation and MM invasiveness, we restored merlin expression in NF2-deficient MM cells. Re-expression of merlin markedly inhibited cell motility, spreading and invasiveness, properties connected with the malignant phenotype of MM cells. To test directly whether merlin inactivation promotes invasion in a nonmalignant system, we used small interfering RNA to silence Nf2 in mouse embryonic fibroblasts (MEFs) and found that downregulation of merlin resulted in enhanced cell spreading and invasion. To delineate signaling events connected with this phenotype, we investigated the effect of merlin expression on focal adhesion kinase (FAK), a key component of cellular pathways affecting migration and invasion. Expression of merlin attenuated FAK phosphorylation at the critical phosphorylation site Tyr397 and disrupted the interaction of FAK with its binding partners Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase. In addition, NF2-null MM cells stably overexpressing FAK showed increased invasiveness, which decreased significantly when merlin expression was restored. Collectively, these findings suggest that merlin inactivation is a critical step in MM pathogenesis and is related, at least in part, with upregulation of FAK activity.     

EphA4 receptor, overexpressed in pancreatic ductal adenocarcinoma, promotes cancer cell growth

To isolate novel diagnostic markers and drug targets for pancreatic ductal adenocarcinoma (PDAC), we previously performed expression profile analysis of PDAC cells using a genome-wide cDNA microarray combined with laser microdissection. Among dozens of up-regulated genes identified in PDAC cells, we herein focused on one tyrosine kinase receptor, Eph receptor A4 (EphA4), as a molecular target for PDAC therapy. Immunohistochemical analysis validated EphA4 overexpression in approximately half of the PDAC tissues. To investigate its biological function in PDAC cells, we knocked down EphA4 expression by siRNA, which drastically attenuated PDAC cell viability. In concordance with the siRNA experiment, PDAC-derivative cells that were designed to constitutively express exogenous EphA4 showed a more rapid growth rate than cells transfected with mock vector, suggesting a growth-promoting effect of EphA4 on PDAC cells. Furthermore, the expression analysis for ephrin ligand family members indicated the coexistence of ephrinA3 ligand in PDAC cells with EphA4 receptor, and knockdown of ephrinA3 by siRNA also attenuated PDAC cell viability. These results suggest that the EphA4-ephrinA3 pathway is likely to be a promising molecular target for pancreatic cancer therapy.     

Increased expression of tissue transglutaminase in pancreatic ductal adenocarcinoma and its implications in drug resistance and metastasis

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases and is virtually incurable. The molecular mechanisms that contribute to the intrinsic resistance of PDAC to various anticancer therapies are not well understood. Recently, we have observed that several drug-resistant and metastatic tumors and tumor cell lines expressed elevated levels of tissue transglutaminase (TG2). Because PDAC exhibits inherent resistance to various drugs, we determined the constitutive expression of TG2 in 75 PDAC and 12 PDAC cell lines. Our results showed that 42 of 75 (56%) PDAC tumor samples expressed higher basal levels of TG2 compared with the normal pancreatic ducts [odds ratio (OR), 2.439; P = 0.012]. The increased expression of TG2 in PDAC was strongly associated with nodal metastasis (OR, 3.400; P = 0.017) and lymphovascular invasion (OR, 3.055; P = 0.045). Increased expression of TG2 was also evident in all 12 cell lines examined. The elevated expression of TG2 in PDAC cell lines was associated with gemcitabine resistance and increased invasive potential. Overexpression of catalytically active or inactive (C(277)S mutant) TG2 induced focal adhesion kinase (FAK) activation and augmented invasive functions in the BxPC-3 cell line. Conversely, down-regulation of TG2 by small interfering RNA attenuated FAK phosphorylation. Immunoprecipitation and confocal microscopy data revealed that TG2 was associated with FAK protein in PDAC cells. The activated FAK colocalized with TG2 at focal adhesion points. These results show for the first time that elevated expression of TG2 can induce constitutive activation of FAK and thus may contribute to the development of drug resistance and invasive phenotypes in PDAC.     

Role of focal adhesion kinase and phosphatidylinositol 3'-kinase in integrin fibronectin receptor-mediated, matrix metalloproteinase-1-dependent invasion by metastatic prostate cancer cells

alpha(5)beta(1) Integrin interacts with the PHSRN sequence of plasma fibronectin, causing constitutive invasion by human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as in vitro invasion substrates. Immunoassays were employed to dissect the roles of focal adhesion kinase (FAK), phosphatidylinositol 3'-kinase (PI3K), and protein kinase Cdelta (PKC delta) in alpha(5)beta(1)-mediated, matrix metalloproteinase-1 (MMP-1)-dependent invasion by metastatic human DU 145 prostate cancer cells. We found that a peptide composed of the PHSRN sequence induced rapid FAK phosphorylation at Tyr(397) (Y397), a site whose phosphorylation is associated with kinase activation. The technique of RNA silencing [small interfering RNA (siRNA)] confirmed the role of FAK in PHSRN-induced invasion. PHSRN also induced the association of the p85-regulatory subunit of PI3K with FAK at a time corresponding to FAK phosphorylation and activation, and maximal PI3K activity occurred at this same time. The necessity of PI3K activity in both PHSRN-induced invasion and MMP-1 expression was confirmed by using specific PI3K inhibitors. By employing a specific inhibitor, Rottlerin, and by using siRNA, we also found that PKC delta, a PI3K substrate found in focal adhesions, functions in PHSRN-induced invasion. In addition, the induction of MMP-1 in PHSRN-treated DU 145 cells was shown by immunoblotting, and the role of MMP-1 in PHSRN-induced invasion was confirmed by the use of blocking anti-MMP-1 monoclonal antibody. Finally, a close temporal correspondence was observed between PHSRN-induced invasion and PHSRN-induced MMP-1 activity in DU 145 cells.     

Human pancreatic tumor cells are sensitized to ionizing radiation by knockdown of caveolin-1

Caveolin-1 (Cav-1) is an integral transmembrane protein and a critical component in interactions of integrin receptors with cytoskeleton-associated and signaling molecules. Since integrin-mediated cell adhesion generates signals conferring radiation resistance, we examined the effects of small interfering RNA-mediated knockdown of Cav-1 alone or in combination with beta1-integrin or focal adhesion kinase (FAK) on radiation survival and proliferation of pancreatic carcinoma cell lines. Irradiation induced Cav-1 expression in PATU8902, MiaPaCa2 and Panc1 cell lines. The cell lines showed significant radiosensitization after knockdown of Cav-1, beta1-integrin or FAK and cholesterol depletion by beta-cyclodextrin relative to nonspecific controls. Under knockdown conditions, proliferation of non-irradiated and irradiated cells was significantly attenuated relative to controls. These findings correlated with changes in expression or phosphorylation of Akt, glycogen synthase kinase 3beta, Paxillin, Src, c-Jun N-terminal kinase and mitogen-activated protein kinase. Analysis of DNA microarray data revealed a Cav-1 overexpression in a subset of pancreatic ductal adenocarcinoma samples. The data presented show, for the first time, that disruption of interactions of Cav-1 with beta1-integrin or FAK affects radiation survival and proliferation of pancreatic carcinoma cells and suggest that Cav-1 is critical to these processes. These results indicate that strategies targeting Cav-1 may be useful as an approach to improve conventional therapies, including radiotherapy, for pancreatic cancer.     

The catalytically defective receptor protein tyrosine kinase EphA10 promotes tumorigenesis in pancreatic cancer cells

EphA10 (erythropoietin‐producing hepatocellular carcinoma receptor A10) is a catalytically defective receptor protein tyrosine kinase in the ephrin receptor family. Although EphA10 is involved in the malignancy of some types of cancer, its role as an oncogene has not been extensively studied. Here, we investigated the influence of EphA10 on the tumorigenic potential of pancreatic cancer cells. Analysis of expression profiles from The Cancer Genome Atlas confirmed that EphA10 was elevated and higher in tumor tissues than in normal tissues in some cancer types, including pancreatic cancer. EphA10 silencing reduced the proliferation, migration, and adhesion of MIA PaCa‐2 and AsPC‐1 pancreatic cancer cells. These effects were reversed by overexpression of EphA10 in MIA PaCa‐2 cells. Importantly, overexpression and silencing of EphA10 respectively increased and decreased the weight, volume, and number of Ki‐67‐positive proliferating cells in MIA PaCa‐2 xenograft tumors. Further, EphA10 expression was positively correlated with invasion and gelatin degradation in MIA PaCa‐2 cells. Moreover, overexpression of EphA10 enhanced the expression and secretion of MMP‐9 in MIA PaCa‐2 cells and increased the expression of MMP‐9 and the vascular density in xenograft tumors. Finally, expression of EphA10 increased the phosphorylation of ERK, JNK, AKT, FAK, and NF‐κB, which are important for cell proliferation, survival, adhesion, migration, and invasion. Therefore, we suggest that EphA10 plays a pivotal role in the tumorigenesis of pancreatic epithelial cells and is a novel therapeutic target for pancreatic cancer.