MINDIN secretion by prostate tumors induces premetastatic ...
W1974 CDx2 Induces Icam2 Expression and Promotes In Vitro Angiogenesis
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W1970 Increased Extracellular Pressure Enhances Cancer Cell β1-Integrin Binding Affinity Through Phosphorylation of β1-Integrin At Threonine 788/789 David H. Craig, Yanzhang Wei, Marc D. Basson Exposure to increased extracellular pressure activates intracellular signals that stimulate cancer cell adhesion to matrix proteins and endothelial cell monolayers In Vitro, and surgical wounds In Vivo by a β1-integrin-dependent mechanism. Such increased adhesiveness may contribute to the metastatic potential of cancer cells exposed to increased pressure during vascular and lymphatic transit, in the tumor microenvironment, or through surgical manip- ulation and postoperative bowel edema. We sought to assess whether pressure-induced cell adhesion is mediated by changes in β1-integrin binding affinity or avidity and whether these changes are phosphorylation-dependent. 15 mmHg increased pressure was applied for 30 minutes to suspended cells using a temperature- and pressure-controlled box. We used a flow cytometric assay to quantify changes in integrin affinity and clustering by measuring differences in binding between single Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab')2 multimeric complexes. We then assessed pressure-stimulated phosphorylation of β1-integrin S785 and T788/9 residues in human SW620 colon cancer cells and primary malignant colonocytes. Finally, we employed GD25 β1-integrin null murine fibroblasts stably trans- fected with either wild-type β1-integrin, S785A, or T788/9A phosphorylation site mutants to assess the dependence of pressure-stimulated cell adhesion on β1-integrin site-specific phosphorylation. RGD-Fc, but not RGD-Fc-F(ab')2, binding to SW620 cells was significantly increased (23±4%; p<0.02; n=4) under elevated pressure, suggesting pressure modulates β1-integrin affinity, but fails to induce integrin clustering. Increased pressure stimulated β1-integrin phosphorylation at both S785 (31±7%; p<0.02; n=5) and T788/9 (28±5%; p<0.01; n=5) residues in SW620 cells. Primary human colon cancer cells responded similarly. GD25 β1-integrin wild-type cells displayed a 30±8% (p<0.02; n=6) increase in adhesion to collagen I under elevated pressure, an effect absent in GD25 β1-integrin null cells (n=6). GD25 cells expressing the S785A β1-integrin phosphorylation mutant also displayed a 19±3% (p<0.01; n=6) increase in adhesion under pressure. However, expression of T788/ 9A β1-integrin completely blocked this effect (n=6). These results suggest that the observed increase in cell adhesion accompanying exposure to elevated pressure reflects enhanced β1- integrin affinity and requires phosphorylation of the cytoplasmic tail of β1-integrin at T788/ 9. The β1-integrin T788/9 phosphorylation site may offer a unique therapeutic target for the modulation of pressure-stimulated cell adhesion and subsequent tumor metastasis. W1971 Nitric Oxide Mediates the Warburg Effect in Gastric Epithelial and Cancer Cells Hirofumi Matsui, Yumiko Nagano, Osamu Shimokawa, Tsuyoshi Kaneko, Jumpei Udo, Takashi Mamiya, Kanho Rai, Ichinosuke Hyodo, Kozi Nakai [Backgrounds and aims]: O. Warburg hypothesized in 1931 that cancer cells preferentially utilize glycolytic pathways for energy generation while down-regulating their aerobic respirat- ory activity. Recent molecular researches revealed the mechanisms behind the decreased aerobic, mitochondrial respiratory activity in cancer cells. Iron-sulfur (Fe-S) clusters are essential components in mitochondrial electron transportation complexes. On Nitric Oxide (NO) treatment, Fe-S cluster is reportedly inactivated and form Dinitorosyl-iron complex (DNIC), resulting in the decreased mitochondrial phosphorylation activity. We hypothesized that the increased intracellular NO in cancer cells may inactivate the Fe-S clusters and attenuate the mitochondrial phosphorylation activity. Here we compared the levels of NO concentrations, DNIC formation, and oxygen consumptions between the gastric epithelial cells and their chemical carcinogen-induced transformed cells, and the results suggested the important role of NO in Warburg effect in cancer cells. [Methods]: Gastric cancer-derived AGS and MKN-45 cells, gastric epithelial-derived RGM-1 cells, and their mutant RGK-36 cells, which had tumorigenetic potential and were regarded as cancerous cells, were used. The oxygen consumption of each cell lines was measured with electrodes. The levels of intracellular NO concentration were measured with fluorescent indicators DAF-2 DA. The DNIC signals of each cellular mitochondrial component with or without exogenous NO treatment were measured with a low temperature Electron Paramagnetic Resonance (EPR). [Results]: The cancerous cells including the transformed RGK-36 cells showed lower oxygen consumption, higher NO concentration than normal RGM-1 cells. Moreover, cancerous cells, not RGM-1 cells, exhibited definite spectra of DNIC, indicating the presence of degraded mitochondrial Fe-S clusters. Exogenous NO treatment induced the stronger DNIC signals in mitochondrial component of RGM-36 cells. [Conclusions] The results of high NO concen- trations, low oxygen consumption, degenerated Fe-S clusters highlight the induction of Warburg effects through the neoplastic transformation in RGK-36 cells. Stronger DINC signals on exogenous NO treatment in cancerous cells suggested NO mediated the phenomenon. W1972 COX-Derived PGE2 Modulates Fasl Expression Via the Ep1 Receptor in Colon Cancer Cells. Aileen Houston, Grace P. O'Callaghan, Fergus Shanahan Background: Fas ligand (FasL/CD95L) is a member of the tumor necrosis factor superfamily that triggers apoptotic cell death following ligation to its receptor, Fas (CD95/APO-1), on sensitive cells. Studies have shown that FasL is expressed in a variety of human cancers, and have strongly implicated tumor-expressed FasL as a major inhibitor of the anti-tumor immune response. Thus, inhibiting FasL expression in tumors may have therapeutic potential, due to an improved anti-tumor immune response. However, little is known about the mechanisms that regulate FasL expression in tumors. Cyclooxygenase-2 (COX-2) and its principal metabolite prostaglandin E 2 (PGE 2 ) have been shown to play an important role in colon carcinogenesis. Aim: To investigate if the COX signaling pathway, and in particular prostaglandin E 2 (PGE 2 ), plays a role in the upregulation of FasL expression in colon cancer. Methods: COX-1 and COX-2 expression was blocked in HT29 and HCA-7 colon tumor cells using both RNA interference (RNAi) and specific COX-1 and COX-2 inhibitors. FasL A-745 AGA Abstracts expression was assessed by real-time RT-PCR and Western blotting. Jurkat cell viability following co-culture with colon tumor cells was assessed by resazurin reduction. In a panel of human colon adenocarcinomas, expression of COX-2 and FasL was detected by immunohistochemistry. Results: Suppression of either COX-1 or COX-2 by RNA interference (RNAi) in HT29 and HCA-7 colon tumor cells reduced FasL expression at both the mRNA and protein level. Conversely, FasL expression was upregulated by PGE 2 and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. This effect was specific for FasL and did not affect other members of the TNF superfamily. This signal was transduced via the EP1 receptor in a PKC and p38 MAPK-dependent manner. Finally, semi-quantitative immunostaining of human colon adenocarcinoma tissue sections revealed a strong positive correlation between FasL and COX-2 expression in tumour tissue (r=0.722; p<0.0001), demonstrating that these observations may have clinical significance. Conclusion: Collect- ively, these findings support a role for PGE 2 as a critical mediator of FasL expression in colon tumor cells. Targeting the PGE 2 - FasL signaling pathway may aid in the development of new therapeutic strategies to both prevent and treat this malignant disease. This work is supported by the Health Research Board, Ireland. W1973 Constitutive Activation of Ikk/Nf-κB in Colorectal Cancer Cells Plays Critical Role in Tumor Growth and Angiogenesis Kei Sakamoto, Shin Maeda, Yohko Hikiba, Hayato Nakagawa, Wataru Shibata, Ayako Yanai, Keiji Ogura, Masao Omata Introduction: Nuclear factor kappa B (NF-κB) is an important transcriptional factor that controls various biological processes such as inflammation, cell cycle and cell survival. Constitutive activation of NF-κB has been noted in many tumor types including colorectal cancer. However, the exact role of constitutive NF-κB activation in colorectal cancer is still unclear. Method: To evaluate constitutive NF-κB activation, EMSA was performed in various colorectal cancer cell lines. To inhibit the activation of the NF-κB, we established cancer cells with stable knockdown of IKKγ (NEMO) which is the regulatory subunit of the IKK complex and is involved in controlling NF-κB activation by using RNA interference. Cell growth and apoptosis were evaluated in wild-type (WT) and knocked down (KD) cells. Cytokines and chemokine expression were also checked by protein array analysis. To deter- mine an involvement of angiogenesis, HUVEC was incubated in the supernatant of WT and KD cells and counted the numbers of branch point, which is a phenotypical change of angiogenesis. By subcutaneously transplantation of cells into BALB/c nude mice, tumor sizes and vascularity were analyzed. Result: Constitutive NF-κB DNA binding activity was increased in about 50% colorectal cancer cell lines. We established IKKγ knockdown cells in LoVo and DLD1 which showed strong constitutive NF-κB activation. In Vitro , cell proliferation was not changed between WT and KD cells. TNFα-mediated apoptosis was increased in KD cells relative to WT cells. By the array analysis, IL-8 and MCP-1, which were reported to be strongly regulated by NF-κB activation, were increased in LoVo and DLD1 compared to other cell lines without constitutive NF-κB activation. KD cells secreted significantly smaller amounts of IL-8 and MCP1 compared with WT cells. As IL-8 and MCP-1 were reported to be angiogenic factors, we evaluated the angiogenesis by incubation HUVEC in the supernatant of WT and KD cells. HUVEC incubated in WT supernatant showed more branch points than in KD supernatant. Although IL-8 and/or MCP-1 knocked down cells showed less angiogenesis compared with WT cells, those cells induced more angiogenesis relative to KD cells, suggesting that certain NF-κB-regulated factors other than IL-8 and MCP-1 may play a role for angiogenesis. In Vivo, tumor expansion was suppressed to 25 % in LoVo KD cells. Tumor vascularity determined by immunohistochemistry was significantly suppressed in KD cells. Conclusion: NF-κB inhibition may become an attractive target for the treatment of colorectal cancer. W1974 CDx2 Induces Icam2 Expression and Promotes In Vitro Angiogenesis Sang Chun, Kyunghee Burkitt, John P. Lynch, Marcia R. Cruz-Correa, Long H. Dang, Duyen Dang INTRODUCTION: CDX2 is a homeobox transcription factor that is important for the estab- lishment and maintenance of intestinal epithelial cells. CDX2 potently induces epithelial cell adhesion and colony compaction. Microarray analysis in our lab identified ICAM2 (intercellular adhesion molecule 2) as a putative CDX2 target gene. ICAM2 is a cell surface adhesion protein expressed on many cells, including endothelial cells. One function of ICAM2 is to promote homophilic cell-cell interactions and angiogenesis. AIM: To determine the effects of CDX2 on ICAM2 expression and function. METHODS: Three pairs of human colon cancer cell lines were used. LOVO cells express high CDX2 and LOVO CDX2-/- cells do not express CDX2 due to somatic disruption. COLO205 do not express CDX2 and COLO205 CDX2+ cells overexpress CDX2. HCT116 cells express low levels of CDX2 and HCT116 CDX2+ cells overexpress CDX2. CDX2 and ICAM2 expression were determined by RT-PCR and western blot analyses. The effects of CDX2 and ICAM2 on colon cancer cell-endothelial cell adhesion was determined by plating human umbilical vein endothelial cells (HUVEC) cells to confluence, then incubating with Calcein AM labeled colon cancer cells. The effects of CDX2 and ICAM2 on endothelial tube formation was determined by co-culture of HUVEC and colon cancer cells on ECMatrix. To block the effects of ICAM2, ICAM2 monoclonal antibodies (5-10 uM) were added to cultures. Last, CDX2 and ICAM2 expression were measured by RT-PCR in 38 primary colon cancers and their adjacent normal mucosa. RESULTS: In all three cell lines, CDX2 induced the expression of ICAM2 by ~3.5- fold. ICAM2 blockade did not affect colony formation when cancer cells were cultured alone In Vitro. CDX2 expression increased colon cancer cells adhesion to HUVEC endothelial cells, which was partially reversed by ICAM2 blockade. Co-cultures of HUVEC cells with CDX2 expressing cells developed into a complex capillary network of branching cords and tubules, which resembled a mesh-like structure with multiple polygons. Either ICAM2 blockade, or co-culture of HUVEC cells with CDX2-negative cells, led to the formation of a simpler capillary network, with thinner capillary cords and less closed polygons. In the primary tumors, CDX2 and ICAM2 expressions were increased and positively correlated. CONCLU- SIONS: These data show, for the first time, that ICAM2 is expressed in colon cancer, and AGA Abstracts
Transcript of W1974 CDx2 Induces Icam2 Expression and Promotes In Vitro Angiogenesis
W1970
Increased Extracellular Pressure Enhances Cancer Cell β1-Integrin BindingAffinity Through Phosphorylation of β1-Integrin At Threonine 788/789David H. Craig, Yanzhang Wei, Marc D. Basson
Exposure to increased extracellular pressure activates intracellular signals that stimulatecancer cell adhesion to matrix proteins and endothelial cell monolayers In Vitro, and surgicalwounds In Vivo by a β1-integrin-dependent mechanism. Such increased adhesiveness maycontribute to the metastatic potential of cancer cells exposed to increased pressure duringvascular and lymphatic transit, in the tumor microenvironment, or through surgical manip-ulation and postoperative bowel edema. We sought to assess whether pressure-induced celladhesion is mediated by changes in β1-integrin binding affinity or avidity and whether thesechanges are phosphorylation-dependent. 15 mmHg increased pressure was applied for 30minutes to suspended cells using a temperature- and pressure-controlled box. We used aflow cytometric assay to quantify changes in integrin affinity and clustering by measuringdifferences in binding between single Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab')2multimeric complexes. We then assessed pressure-stimulated phosphorylation of β1-integrinS785 and T788/9 residues in human SW620 colon cancer cells and primary malignantcolonocytes. Finally, we employed GD25 β1-integrin null murine fibroblasts stably trans-fected with either wild-type β1-integrin, S785A, or T788/9A phosphorylation site mutantsto assess the dependence of pressure-stimulated cell adhesion on β1-integrin site-specificphosphorylation. RGD-Fc, but not RGD-Fc-F(ab')2, binding to SW620 cells was significantlyincreased (23±4%; p<0.02; n=4) under elevated pressure, suggesting pressure modulatesβ1-integrin affinity, but fails to induce integrin clustering. Increased pressure stimulatedβ1-integrin phosphorylation at both S785 (31±7%; p<0.02; n=5) and T788/9 (28±5%;p<0.01; n=5) residues in SW620 cells. Primary human colon cancer cells responded similarly.GD25 β1-integrin wild-type cells displayed a 30±8% (p<0.02; n=6) increase in adhesion tocollagen I under elevated pressure, an effect absent in GD25 β1-integrin null cells (n=6).GD25 cells expressing the S785A β1-integrin phosphorylation mutant also displayed a19±3% (p<0.01; n=6) increase in adhesion under pressure. However, expression of T788/9A β1-integrin completely blocked this effect (n=6). These results suggest that the observedincrease in cell adhesion accompanying exposure to elevated pressure reflects enhanced β1-integrin affinity and requires phosphorylation of the cytoplasmic tail of β1-integrin at T788/9. The β1-integrin T788/9 phosphorylation site may offer a unique therapeutic target forthe modulation of pressure-stimulated cell adhesion and subsequent tumor metastasis.
W1971
Nitric Oxide Mediates the Warburg Effect in Gastric Epithelial and CancerCellsHirofumi Matsui, Yumiko Nagano, Osamu Shimokawa, Tsuyoshi Kaneko, Jumpei Udo,Takashi Mamiya, Kanho Rai, Ichinosuke Hyodo, Kozi Nakai
[Backgrounds and aims]: O. Warburg hypothesized in 1931 that cancer cells preferentiallyutilize glycolytic pathways for energy generation while down-regulating their aerobic respirat-ory activity. Recent molecular researches revealed the mechanisms behind the decreasedaerobic, mitochondrial respiratory activity in cancer cells. Iron-sulfur (Fe-S) clusters areessential components in mitochondrial electron transportation complexes. On Nitric Oxide(NO) treatment, Fe-S cluster is reportedly inactivated and form Dinitorosyl-iron complex(DNIC), resulting in the decreased mitochondrial phosphorylation activity. We hypothesizedthat the increased intracellular NO in cancer cells may inactivate the Fe-S clusters andattenuate the mitochondrial phosphorylation activity. Here we compared the levels of NOconcentrations, DNIC formation, and oxygen consumptions between the gastric epithelialcells and their chemical carcinogen-induced transformed cells, and the results suggested theimportant role of NO in Warburg effect in cancer cells. [Methods]: Gastric cancer-derivedAGS and MKN-45 cells, gastric epithelial-derived RGM-1 cells, and their mutant RGK-36cells, which had tumorigenetic potential and were regarded as cancerous cells, were used.The oxygen consumption of each cell lines was measured with electrodes. The levels ofintracellular NO concentration were measured with fluorescent indicators DAF-2 DA. TheDNIC signals of each cellular mitochondrial component with or without exogenous NOtreatment were measured with a low temperature Electron Paramagnetic Resonance (EPR).[Results]: The cancerous cells including the transformed RGK-36 cells showed lower oxygenconsumption, higher NO concentration than normal RGM-1 cells. Moreover, cancerouscells, not RGM-1 cells, exhibited definite spectra of DNIC, indicating the presence of degradedmitochondrial Fe-S clusters. Exogenous NO treatment induced the stronger DNIC signalsin mitochondrial component of RGM-36 cells. [Conclusions] The results of high NO concen-trations, low oxygen consumption, degenerated Fe-S clusters highlight the induction ofWarburg effects through the neoplastic transformation in RGK-36 cells. Stronger DINC signalson exogenous NO treatment in cancerous cells suggested NO mediated the phenomenon.
W1972
COX-Derived PGE2 Modulates Fasl Expression Via the Ep1 Receptor in ColonCancer Cells.Aileen Houston, Grace P. O'Callaghan, Fergus Shanahan
Background: Fas ligand (FasL/CD95L) is a member of the tumor necrosis factor superfamilythat triggers apoptotic cell death following ligation to its receptor, Fas (CD95/APO-1), onsensitive cells. Studies have shown that FasL is expressed in a variety of human cancers,and have strongly implicated tumor-expressed FasL as a major inhibitor of the anti-tumorimmune response. Thus, inhibiting FasL expression in tumors may have therapeutic potential,due to an improved anti-tumor immune response. However, little is known about themechanisms that regulate FasL expression in tumors. Cyclooxygenase-2 (COX-2) and itsprincipal metabolite prostaglandin E2 (PGE2) have been shown to play an important rolein colon carcinogenesis. Aim: To investigate if the COX signaling pathway, and in particularprostaglandin E2 (PGE2), plays a role in the upregulation of FasL expression in colon cancer.Methods: COX-1 and COX-2 expression was blocked in HT29 and HCA-7 colon tumorcells using both RNA interference (RNAi) and specific COX-1 and COX-2 inhibitors. FasL
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expression was assessed by real-time RT-PCR and Western blotting. Jurkat cell viabilityfollowing co-culture with colon tumor cells was assessed by resazurin reduction. In apanel of human colon adenocarcinomas, expression of COX-2 and FasL was detected byimmunohistochemistry. Results: Suppression of either COX-1 or COX-2 by RNA interference(RNAi) in HT29 and HCA-7 colon tumor cells reduced FasL expression at both the mRNAand protein level. Conversely, FasL expression was upregulated by PGE2 and these cellsshowed increased cytotoxicity against Fas-sensitive Jurkat T cells. This effect was specificfor FasL and did not affect other members of the TNF superfamily. This signal was transducedvia the EP1 receptor in a PKC and p38 MAPK-dependent manner. Finally, semi-quantitativeimmunostaining of human colon adenocarcinoma tissue sections revealed a strong positivecorrelation between FasL and COX-2 expression in tumour tissue (r=0.722; p<0.0001),demonstrating that these observations may have clinical significance. Conclusion: Collect-ively, these findings support a role for PGE2 as a critical mediator of FasL expression incolon tumor cells. Targeting the PGE2 - FasL signaling pathway may aid in the developmentof new therapeutic strategies to both prevent and treat this malignant disease. This work issupported by the Health Research Board, Ireland.
W1973
Constitutive Activation of Ikk/Nf-κB in Colorectal Cancer Cells Plays CriticalRole in Tumor Growth and AngiogenesisKei Sakamoto, Shin Maeda, Yohko Hikiba, Hayato Nakagawa, Wataru Shibata, AyakoYanai, Keiji Ogura, Masao Omata
Introduction: Nuclear factor kappa B (NF-κB) is an important transcriptional factor thatcontrols various biological processes such as inflammation, cell cycle and cell survival.Constitutive activation of NF-κB has been noted in many tumor types including colorectalcancer. However, the exact role of constitutive NF-κB activation in colorectal cancer is stillunclear. Method: To evaluate constitutive NF-κB activation, EMSA was performed in variouscolorectal cancer cell lines. To inhibit the activation of the NF-κB, we established cancercells with stable knockdown of IKKγ (NEMO) which is the regulatory subunit of the IKKcomplex and is involved in controlling NF-κB activation by using RNA interference. Cellgrowth and apoptosis were evaluated in wild-type (WT) and knocked down (KD) cells.Cytokines and chemokine expression were also checked by protein array analysis. To deter-mine an involvement of angiogenesis, HUVEC was incubated in the supernatant of WT andKD cells and counted the numbers of branch point, which is a phenotypical change ofangiogenesis. By subcutaneously transplantation of cells into BALB/c nude mice, tumor sizesand vascularity were analyzed. Result: Constitutive NF-κB DNA binding activity was increasedin about 50% colorectal cancer cell lines. We established IKKγ knockdown cells in LoVoand DLD1 which showed strong constitutive NF-κB activation. In Vitro , cell proliferationwas not changed between WT and KD cells. TNFα-mediated apoptosis was increased inKD cells relative to WT cells. By the array analysis, IL-8 and MCP-1, which were reportedto be strongly regulated by NF-κB activation, were increased in LoVo and DLD1 comparedto other cell lines without constitutive NF-κB activation. KD cells secreted significantlysmaller amounts of IL-8 and MCP1 compared with WT cells. As IL-8 and MCP-1 werereported to be angiogenic factors, we evaluated the angiogenesis by incubation HUVEC inthe supernatant of WT and KD cells. HUVEC incubated in WT supernatant showed morebranch points than in KD supernatant. Although IL-8 and/or MCP-1 knocked down cellsshowed less angiogenesis compared with WT cells, those cells induced more angiogenesisrelative to KD cells, suggesting that certain NF-κB-regulated factors other than IL-8 andMCP-1 may play a role for angiogenesis. In Vivo, tumor expansion was suppressed to 25 %in LoVo KD cells. Tumor vascularity determined by immunohistochemistry was significantlysuppressed in KD cells. Conclusion: NF-κB inhibition may become an attractive target forthe treatment of colorectal cancer.
W1974
CDx2 Induces Icam2 Expression and Promotes In Vitro AngiogenesisSang Chun, Kyunghee Burkitt, John P. Lynch, Marcia R. Cruz-Correa, Long H. Dang,Duyen Dang
INTRODUCTION: CDX2 is a homeobox transcription factor that is important for the estab-lishment and maintenance of intestinal epithelial cells. CDX2 potently induces epithelialcell adhesion and colony compaction. Microarray analysis in our lab identified ICAM2(intercellular adhesion molecule 2) as a putative CDX2 target gene. ICAM2 is a cell surfaceadhesion protein expressed on many cells, including endothelial cells. One function ofICAM2 is to promote homophilic cell-cell interactions and angiogenesis. AIM: To determinethe effects of CDX2 on ICAM2 expression and function. METHODS: Three pairs of humancolon cancer cell lines were used. LOVO cells express high CDX2 and LOVO CDX2-/- cellsdo not express CDX2 due to somatic disruption. COLO205 do not express CDX2 andCOLO205 CDX2+ cells overexpress CDX2. HCT116 cells express low levels of CDX2 andHCT116 CDX2+ cells overexpress CDX2. CDX2 and ICAM2 expression were determinedby RT-PCR and western blot analyses. The effects of CDX2 and ICAM2 on colon cancercell-endothelial cell adhesion was determined by plating human umbilical vein endothelialcells (HUVEC) cells to confluence, then incubating with Calcein AM labeled colon cancercells. The effects of CDX2 and ICAM2 on endothelial tube formation was determined byco-culture of HUVEC and colon cancer cells on ECMatrix. To block the effects of ICAM2,ICAM2 monoclonal antibodies (5-10 uM) were added to cultures. Last, CDX2 and ICAM2expression were measured by RT-PCR in 38 primary colon cancers and their adjacent normalmucosa. RESULTS: In all three cell lines, CDX2 induced the expression of ICAM2 by ~3.5-fold. ICAM2 blockade did not affect colony formation when cancer cells were cultured aloneIn Vitro. CDX2 expression increased colon cancer cells adhesion to HUVEC endothelial cells,which was partially reversed by ICAM2 blockade. Co-cultures of HUVEC cells with CDX2expressing cells developed into a complex capillary network of branching cords and tubules,which resembled a mesh-like structure with multiple polygons. Either ICAM2 blockade, orco-culture of HUVEC cells with CDX2-negative cells, led to the formation of a simplercapillary network, with thinner capillary cords and less closed polygons. In the primarytumors, CDX2 and ICAM2 expressions were increased and positively correlated. CONCLU-SIONS: These data show, for the first time, that ICAM2 is expressed in colon cancer, and
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sCDX2 regulates ICAM2 expression. The effects of CDX2 induction of ICAM2 are not cell-autonomous, as In Vitro colony formation is not affected. However, CDX2 induction ofICAM2 promotes cancer cell-endothelial cell interactions and angiogenesis.
W1975
Genetic Inhibition of Telomerase Leads to Induction of ALT in ImmortalizedHuman Esophageal Epithelial Cells and in Generated Human SquamousCancer CellsAngela Queisser, Michaela Thaler, Alexander von Werder, Eva Egenter, Steffen Heeg, NinaHirt, Heike Kunert, Sarah Hauss, Hideki Harada, Hiroshi Nakagawa, Shang Li, ElisabethH. Blackburn, Hubert E. Blum, Oliver G. Opitz
Introduction: During malignant transformation telomere maintenance is important forimmortalization. Telomere maintenance is either mediated through activation of the enzymetelomerase, which contains an RNA-template (hTER) and the core protein (hTERT) orthrough a recombination based alternative mechanism (ALT). Little is known about theregulation of these two mechanisms in a single cell. We investigated whether ALT can beinduced in genetically defined immortalized cells or malignant transformed cancer cells byspecific genetic telomerase inhibitors. Methods: We generated immortalized human eso-phageal epithelial cells overexpressing Cyclin D1 or hTERT, respectively (EPC D1 and EPChTERT) as well as human oral squamous cancer cells by overexpression of Cyclin D1,d.n.p53, EGFR and c-myc (OKF6 D1/d.n.p53/EGFR/c-myc) using retroviral transduction.To genetically inhibit telomerase, all cell types were transduced with mutated versions ofhTER, anti-hTER, siRNA or a combination of both by lentiviral mediated gene transfer.Transduced cells were sorted by GFP-coexpression. Growth behavior, telomerase activity(TRAP-assay) and telomere length (PFGE-TRF and Q-FISH) were assayed. Indirect immuno-fluorescence for telomere related proteins (APBs) was performed. Results: Overexpressionof MT-hTER, anti-hTER siRNA and the combination of both rapidly inhibits cell growth intransduced immortalized cells but not in transduced generated cancer cells. A reduction oftelomerase activity could be observed in all hTER-inhibited cell types, whereas TRF analysisrevealed telomere elongation, which is characteristic for ALT. Q-FISH analysis suggested aheterogeneous pattern of telomere length on a chromosomal level. In contrast, control cellsdisplayed a robust telomerase activity and a homogenous telomere length in immortalizedas well as in malignant transformed cells. Additionally, in indirect immunofluorescence ALT-associated PML bodies (APBs) were observed in a higher frequency in immortalized hTER-inhibited cells. Summary: Genetically defined immortalized human esophageal epithelialcells and the generated cancer cells are capable to elongate their telomeres using bothtelomere maintenance mechanisms, namely ALT or telomerase activation. ALT can be inducedby the inhibition of telomerase with mutant template telomerase RNA, anti-telomerase siRNAand the combination of both. These findings suggest that immortalized and malignanttransformed cells and eventually all telomerase positive cancer cells, treated with telomeraseinhibitors as potential anti-cancer strategy might find alternative ways to maintain their telom-eres.
W1976
Opposing Effects of Deoxycholic Acid and Ursodeoxycholic Acid On GolgiStructure; Implications for Colon Cancer ProgressionAnne-Marie Byrne, Eilis Foran, Ruchika Sharma, Anthony M. Davies, Dermot P. Kelleher,Aideen Long
Deoxycholic acid (DCA) is a hydrophobic bile acid implicated in colon cancer. We haveobserved that DCA causes fragmentation of the Golgi, an organelle responsible for proteinprocessing in the cell. The Golgi packages and transports proteins by a process calledmembrane fission which involves activation of the enzymes PKCη and PKD. Hyperactivationof this process has important implications as disruption of the Golgi can lead to abnormallyglycosylated proteins which are inherent in malignant cells. The aim of this study was toinvestigate the mechanism of DCA-induced Golgi fragmentation and the effect of UDCA,previously shown to be cytoprotective and antagonistic to DCA, on this process. HCT116colon carcinoma cells were treated with 300 µM DCA and Golgi were visualised using afluorescently labelled Golgi (GM130) antibody. Golgi fragmentation was assessed by HighContent Analysis. DCA was shown to induce Golgi fragmentation in 54.40 ± 1.19% cellscompared to 33.28 ± 2.21% in untreated cells (p<0.05). DCA induced this Golgi fragmenta-tion via activation of the PKCη - PKD pathway as demonstrated by Western blot analysisusing phospho-specific antibodies. Pre-treatment of cells with 300 µM UDCA decreasedDCA-induced Golgi fragmentation (42.87 ± 4.36%, p<0.05). UDCA inhibited DCA-inducedPKD activation and also inhibited Golgi fragmentation induced by constitutively active PKCη.As UDCA has previously been shown to bind the glucocorticoid receptor (GR) we investigatedwhether UDCA-mediated inhibition of DCA-induced Golgi fragmentation was via activationof the GR. UDCA could no longer overcome DCA-induced Golgi fragmentation when cellswere treated with the GR antagonist Mifipristone or following knockdown of the GR usingsiRNA. This demonstrates that UDCA mediates its protective effects via the GR in this system.( F r a g m e n t a t i o n : D C A : 5 4 . 4 0 ± 1 . 1 9 % , U D C A + D C A : 4 2 . 8 7 ± 4 . 3 6 % ,UDCA+DCA+Mifipristone: 57.43 ± 3.68%, UDCA+DCA+ siRNA: 51.66 ± 2.54%). We con-clude that DCA-induced Golgi fragmentation is due to overactivation of the membranefission process via activation of PKCη and phosphorylation of PKD. These effects werereversed by UDCA which acts through the GR. This study identifies DCA as the firstphysiological inducer of Golgi fragmentation, and suggests UDCA may be used as a chemop-reventative agent. This study also gives an insight into mechanisms of bile-acid signallingwhich may be involved in neoplastic progression in the colon.
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W1977
Trastuzumab Induced CTL Responses Against Esophageal Cancer AreInhibited By Down-Regulation of the Antigen Processing MachineryComponent Tap2Francesca Milano, Agnieszka M. Rygiel, Wytske Westra, Kausilia K. Krishnadath
Introduction: Esophageal adenocarcinoma (EAC) is a disease with an extremely poor pro-gnosis. In a previous study we demonstrated that Dendritic Cell (DC) therapy is a promisingtreatment option for EAC. We earlier found high HER-2 gene amplification in up to 50%EACs. HER-2-derived peptides are naturally processed as cytotoxic T cells (CTL) epitopesthat can be recognized by CTL. The humanized antibody against HER-2, Trastuzumab, haspotent inhibitory effects on HER-2 overexpressing tumors. One of the mechanisms of actionof Trastuzumab is the enhancement of the CTL response through improvement of HER-2epitopes presentation via MHC-Class I molecule on tumor cells that may mediate a HER-2 specific CTL response and tumor cell lysis. It is known that deficiencies of componentsof the antigen processing machinery (APM) are responsible for reduced antigen presentationby MHC-Class-I molecules, which is an important immuno-surveillance-escape mechanismof cancers. We previously demonstrated that by treating HER-2 positive EAC cell lines withTrastuzumab, there is enhanced CTL response against the cell line OE33, but this effectwas not observed in the cell line OE19. Aim: to investigate whether down regulation of theAPM is responsible for inhibition of a Trastuzumab mediated CTL response in OE19 andin EAC in general and if so, to investigate whether a deficient APM can be restored byinterferon gamma (INF-gamma). Methods: RT-PCR and immunohistochemistry (IHC) wereapplied to study the expression of the three most important components of the APM:Transporters Associated with antigen Processing 1 and 2 (TAP1 and 2), and Tapasin, inthree EAC cell lines, OE33, OE19 (HER-2 positive) and Bic-1 (HER-2 negative) and intumor biopsies taken from EAC patients. In case of TAP deficiencies, IFN-gamma, knownas a potent inducer of TAP expression, was used to investigate whether TAP deficienciescould be restored. Results: Both at RNA and protein level, expression of TAP1 and Tapasinwere observed in all the three EAC cell lines, and in 88% of the patient tumor biopsies.Interestingly, expression of TAP2 was lost in the EAC cell line OE19, and in 41% of thetumor biopsies. Moreover, by treating the cell lines with IFN-gamma, the TAP2 functioncould be restored in OE19. Conclusions: The unresponsiveness of OE19 to Trastuzumabmediated CTL response may be due to deficient APM. Down regulated expression of TAP2is a frequent event in EAC, and may also determine unresponsiveness of HER-2 positiveEAC patients to Trastuzumab. However, the unresponsiveness to Trastuzumab might beovercome by treatment with INF-gamma that directly restores the APM by up-regulatingTAP2.
W1978
B7-H1 Is Highly Expressed in Gastric Carcinoma and Suppresses Intratumor TCell Immune ResponseZhanju Liu, Shuman Liu
Background & Aims: B7-H1 is expressed in antigen presenting cells, and functions as onekind of co-stimulatory molecules associated with PD-1 expressed in T lymphocytes. Itplays an important role in immune regulation through inhibiting T cell activation anddifferentiation. B7-H1/PD-1 interaction has been found to involve in cancer immune evasionfrom the host immune surveillance system in several malignancies, while the relevance tocarcinogenesis in gastric carcinoma is still elusive. In this study, we investigated the expressionof B7-H1 in human gastric carcinoma and its functional role in regulating T cell activationin anti-tumor immune response. Materials and Methods: B7-H1 expression in gastrictissue and gastric carcinoma cell lines (SGC7901, SGC/VCR, BGC823) was determined byimmunohistochemistry, RT-PCR and flow cytometric analysis. Cytokines were detected byELISA. Results: Immunohistochemical analysis revealed that B7-H1 was highly expressedin 64.4% (47/73) of gastric carcinoma specimens, and PD-1 was present in 43.8% (32/73)of gastric carcinoma specimens examined. However, no B7-H1 and PD-1 expression weredetected in normal gastric tissue by immunohistochemistry. Alpha-naphthyl acetate esterasestaining demonstrated that B7-H1 expression was inversely correlated with tumor infiltrationlymphocyte (TIL) infiltration, particularly CD8+ T cells in gastric carcinoma. B7-H1 mRNAwas also found to be increased expression in all gastric carcinoma tissue compared withhealthy controls (P < 0.05). Additionally, all 3 gastric carcinoma cell lines constitutivelyexpressed B7-H1 mRNA and protein. To elucidate the functional relevance of B7-H1 moleculeto the induction of intra-tumor T cell immune response, T cells (1×106/well) isolated fromgastric carcinoma tissue were stimulated In Vitro with B7-H1 fusion protein (5 µg/ml) inthe presence of immobilized anti-CD3 mAb (5 µg/ml) for 48h. Interestingly, the resultsdemonstrated that T cells isolated from gastric carcinoma tissue produced higher levels ofinterleukin-10 and lower levels of IFN-γ compared with healthy controls (P < 0.05). Conclu-sion: B7-H1 is highly expressed in gastric carcinoma, and closely associated with theinhibition of T-cell anti-tumor activities. This work highlights the important role of B7-H1in immune surveillance to gastric carcinoma growth and represents a novel mechanism bywhich gastric carcinoma cells evade immune recognition and destruction.
W1979
Telomerase Activity and Telomere Length in Lcm Purified Barrett's EsophagealAdenocarcinoma CellsMasood A. Shammas, Aamer Qazi, Ramesh B. Batchu, Jason Y. Wong, Manjula Y. Rao,Christopher S. Bryant, Sanjeev Kumar, Madhu Prasad, Christopher P. Steffes, ImmaculataDe Vivo, David G. Beer, Donald W. Weaver, Raj K. Goyal
INTRODUCTION: The aim of this study was to assess telomere length and telomeraseactivity in normal and Barrett's adenocarcinoma (BEAC) cells purified by laser capturemicrodissection (LCM). METHODS: Epithelial cells were identified according to standardhistopathological criteria and purified by LCM from tissue samples of surgically resectednormal, Barrett's, and BEAC esophagi. Telomerase activity was assessed by an improvedversion of the original Telomeric Repeat Amplification Protocol assay whereas telomere
