bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2264: Lumican as a new target to develop molecularly-targeted drugs for pancreatic cancer
Authors: Yamamoto Tetsushi , Ishiwata Toshiyuki , Matsuda Yoko , Onda Munehiko , Yamahatsu Kazuya , Kawahara Kiyoko , Kawamoto Yoko , Uchida Eiji , Naito Zenya
Published: February 2011

Background: Lumican is a member of a small leucine-rich proteoglycan family and its overexpression has been reported in breast, colorectal, neuroendocrine cell, uterine cervical and pancreatic cancers. The expression of lumican in stromal tissues in breast cancer is associated with a high tumor grade, a low estrogen receptor expression level, and young age. Lumican expression in the cytoplasm in advanced colorectal cancer is correlated with a poor prognosis. Lumican expression was previously reported in pancreatic cancer, but the role of lumican in pancreatic cancer is still not well understood. In this study, we clarified the role of lumican in pancreatic cancer. Methods: Immunohistochemical analysis using anti-lumican antibody was performed in 65 pancreatic cancer patients. Lumican mRNA and protein were examined in pancreatic cancer cell lines (i.e. PANC-1, MIA PaCa-2, KLM-1) by real-time PCR and western blot. Lumican stably transfected PANC-1 cells were prepared. Then, the growth rates in vitro and in vivo were compared with mock cells. The lumican expression of PANC-1 cell was knocked down using siRNA. Then, the growth rate in vitro was compared with the control cell. Results: Immunohistochemically, lumican was localized in the cytoplasm of cancer cells in 33 of 65 patients (51%). Lumican expression in cancer cells did not correlated with clinicopathological factors. However, the survival rate of patients with lumican-positive cancer cells was significantly lower than those with lumican-negative cancer cells in the surgery-only group. Lumican mRNA and the protein were detected in all pancreatic cancer cell lines and PANC-1 cells expressed median level of lumican in the cancer cell lines examined. The growth rate of lumican-transfected PANC-1 cells was significantly higher than that of mock cells both in vitro and in vivo. Moreover, the growth rate of lumican-knocked down PANC-1 cell was significantly lower than that of the control cell. Conclusion: Lumican might regulate pancreatic cancer cell growth and become a new target to develop anti-cancer drug for pancreatic cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2264.
Abstract 2266: Pioglitazone increases tumor progression and metastasis in an orthotopic mouse model of non-small cell lung cancer
Authors: Li Howard Y. , Sorenson Amber , Amin Jay , Middleton Deryck , Weiser-Evans Mary , Nemenoff Raphael A.
Published: February 2011

RATIONALE: Pathways regulating lung cancer metastasis are not well understood. As with most cancers, metastasis depends on critical interactions between tumor cells and the surrounding tumor microenvironment. Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. In humans, a retrospective study of patients treated with thiazolidinediones (TZDs), which are ligands for PPARγ, demonstrated a 33% reduction in risk of developing lung cancer. However, the effect of PPARγ activation on progression and metastasis of established lung tumors has not been clearly established. Our laboratory has developed a mouse model in which murine lung cancer cells directly injected into the lung of syngeneic mice develop secondary pulmonary tumors and metastasize to the mediastinal lymph nodes, the liver, and the brain. The goal of this study was to assess the effects of the TZD pioglitazone on lung cancer progression using this model.

METHODS: Murine non-small cell lung cancer cells stably transfected with firefly luciferase were implanted in the left lung of C57BL/6 wild-type mice. Mice were fed either chow impregnated with pioglitazone (0.05%) or control chow. At various time points following injection, animals were sacrificed and organs were assessed for tumors using ex vivo bioluminescence imaging. Primary tumors were measured with digital calipers, and the number of secondary tumors in the other lung lobes, liver and brain were quantified.

RESULTS: There was no significant difference in the size of the primary tumor between pioglitazone-treated and control mice at one week. However, primary tumors were significantly larger in pioglitazone-treated animals both 16 and 22 days after implantation of cancer cells. The differences in pulmonary metastases were even more impressive. At both 7 and 16 days, pioglitazone-treated mice showed 2-3 times more secondary pulmonary tumors compared to control animals. By day 25-30, 37.5% of mice fed pioglitazone-impregnated chow developed liver metastases, whereas only 17.6% of mice fed normal chow developed liver metastases. Brain metastases were only detected in mice fed chow impregnated with pioglitazone.

CONCLUSION: Treatment of mice with the PPARγ activator pioglitazone unexpectedly accelerates metastasis of established pulmonary tumors. Since selective activation of PPARγ in tumor cells has been shown to inhibit transformed growth and invasiveness, we hypothesize that the metastasis-promoting effects of pioglitazone are mediated through effects on the tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2266.
Abstract 2267: Inhibition of extracellular sulfatase 2 as a possible mechanism to partially explain the anticancer activity of the nitrone OKN007
Authors: Chandru Hema K. , Towner Rheal A. , Floyd Robert A.
Published: February 2011

Sulfatase 2 (Sulf2) is an extracellular enzyme that has endosulfatase activity and catalyzes the removal of 6-O-sulfate groups on glucosamine from subregions of the glycosaminoglycans heparin sulfate. The action of Sulf2 has been shown to alter the binding of protein ligands to heparin which is involved in the binding of growth factors such as VEGF, FGF-2 and TGF to cellular receptors. Studies in experimental models have shown that fully active Sulf2 is important in the development of breast cancer and pancreatic cancer. Several tumors have been shown to be enriched in Sulf2 in comparison to the normal tissues where its level is either absent or present at very low levels. Sulf2 acts as a potentiator for the Wnt signaling and this is considered important in cancer stem cell growth.

We have shown that PBN (alpha-phenyl-tert-butylnitrone) has anti-cancer activity in three experimental models, A) the choline deficiency induced liver cancer model, B) the APCmin model of colon cancer and C) the rat C6 glioma model. We have also shown that 2,4-disulfonyl-alpha-phenyl-tert-butylnitrone (OKN007) has potent anti-cancer activity in the rat C6 glioma model. We considered based on the presence of phenyl sulfonyl groups that the nitrone 2,4-disulfonyl-alpha-phenyl-tert-butylnitrone (OKN007) may act as a competitive inhibitor of Sulf2.

We have found that Sulf2 is secreted into the media of Sulf2 transfected Hek-293 cells as well as several cancer cells, including from the highest to lower levels; MCF-7 breast cancer cells, Hek-293 cells, C6 glioma cells and BxPc-3 pancreatic cancer cells. Utilizing the concentrated media of these cells we have shown that Sulf2 has aryl sulfatase activity against the sulfate ester of 4-methylumbelliferone. We have shown that phenyl sulfonyl compounds suppress Sulf2 activity in an apparent competitive action. For instance, we found that the polysulfonated compound suramin potently suppressed the Sulf2 aryl sulfatase activity and that 2,4-disulfonylphenyl-tert-butylnitrone inhibited Sulf2 activity but less so than suramin. Utilizing concentrated extracellular media of several human cancer cell lines we have shown that Sulf2 activity is decreased by incubation with H2O2. Our results suggest that the anti-cancer activity of OKN007 may in part be explained by its inhibitory action against Sulf1. Research to test this concept is now underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2267.
Abstract 2271: Dabigatran etexilate blocks breast cancer progression in vitro and in a 4T1 breast cancer tumor model in mice
Authors: DeFeo Karen , Hayes Candace S. , Ryn Joanne Van , Gilmour Susan K.
Published: February 2011

Introduction: Coagulation proteases and the generation of thrombin are increased in tumors. Thrombin can modify tumor cell behavior directly through the activation of protease-activated receptors (PAR) or indirectly by generating fibrin matrices.

Aim: To test the hypothesis that dabigatran etexilate (Pradaxa®), an oral direct thrombin inhibitor, can prevent the progression of tumor cells in experimental models.

Materials and Methods: The effect of dabigatran on invasiveness of MDA-MB-231 breast carcinoma cells was evaluated in vitro by invasion across Matrigel-coated membranes and in vivo using a tracheal xenotransplant invasion assay in athymic nude mice. To evaluate the effect of dabigatran on the development of metastatic foci, 4T1 tumor cells were injected orthotopically in the mammary fat pads of syngeneic Balb/c mice. Tumor growth and metastasis was monitored weekly. Dabigatran etexilate was administered twice daily at a dose of 45 mg/kg over 4 weeks in all in vivo models.

Results: Whereas dabigatran had no effect on the proliferation index of cultured tumor cells, it inhibited invasiveness of MDA-MB-231 cells across Matrigel-coated membranes. In vivo evaluation of invasiveness of MDA-MB-231 cells in tracheal xenotransplants in mice administered dabigatran for 3 and 4 weeks demonstrated significantly less penetration of tumor cells through the tracheal wall. Dabigatran treatment had a modest inhibitory effect on growth of the primary 4T1 mammary carcinoma (∼50% reduction in tumor volume at 4 weeks) in syngeneic Balb/c mice, and it reduced both 4T1 tumor cells in the blood and liver micrometastases by 50-60%. Dabigatran treatment resulted in no weight loss in mice.

Conclusion: Oral administration of the direct thrombin inhibitor, dabigatran etexilate, inhibits both invasion and metastasis of malignant breast tumors, suggesting that dabigatran may be beneficial in not only preventing thrombotic events in cancer patients, but also as adjunct therapy to treat malignant tumors. (Supported by funds from Boehringer Ingelheim Pharma)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2271.
Abstract 2278: Tumor microenvironment might be more important than pro-angiogenic chemotactic factors for tumor angiogenesis
Authors: Lee Myungjin , Chang Hyo Won , Nam Hae Yun , Lee Hyangju , Han Myung Woul , Kim Seong Who , Kim Sang Yoon
Published: February 2011

During angiogenesis, many different tumor cytokines have been known to be involved in the regulation of chemotactic endothelial cell migration. Among those, a vascular endothelial growth factor (VEGF) is one of the most essential pro-angiogenic factors and the blockage of VEGF action through competitive inhibition on its receptor or by using of neutralizing antibody such as avastin has been considered as a promising anti-cancer approach to treat several solid tumors. However, despite their ability of releasing VEGF, some cancers show scarce angiogenesis and resistance to avastin treatment.

To explore key factors deciding tumor angiogenesis apart from well-known pro-angiogenic cytokines, we compared the pro-angiogenic capability of the AMC-HN9 head and neck cancer cell line (HN9) which has a low degree of angiogenesis in vivo and are reluctant to avastin treatment with that of the highly angiogenic AMC-HN3 cancer cell line (HN3). Of interest, in vitro cytokine assay of HN9 culture media revealed no difference in the secretion profile of pro-angiogenic cytokines such as VEGF, PlGF, FGF2 and PDGF compared with those of HN3. In addition, HN9 media could strongly promoted tubular formation of HUVECs and migration of fibroblasts in vitro as much as HN3 could. Next, we proceeded to HN9 xenograft co-injected with ECM molecules including fibronectin, collagen III or matrigel. In the co-injected xenografts, density of CD34 positivity significantly increased and the tumor growth exceeded that of HN9 alone-injected ones. Moreover, prominent fibroblast infiltration adjacent to tumor vessels was detected in the co-injected xenografts.

Taken together, these results imply that the formation of extracellular matrix surrounding tumor cells and the presence of activated fibroblasts are pre-requisites and more decisive in angiogenesis than tumor secreted pro-angiogenic cytokine per se.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2278.
Abstract 228: Cucurbitacin-I (JSI-124) induces apoptosis in chronic lymphocytic leukemia (CLL) cells through inhibiting STAT3 activation and XIAP expression
Authors: Ishdorj Ganchimeg
Published: February 2011

Abstract AACR April-2010

Cucurbitacin-I (JSI-124) induces apoptosis in Chronic Lymphocytic Leukemia (CLL) cells through inhibiting STAT3 activation and XIAP expression.

Ganchimeg Ishdorj, James B Johnston and Spencer B Gibson

Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in North America. Current therapies control the disease but eventually the disease recurs and becomes drug resistant. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in cell survival and induces proliferation, whereas deletion of STAT3 results in cell death. Constitutive phosphorylation of STAT3 on tyrosine 705 residues has been found in a number of solid tumors and hematologic malignancies. Unlike other tumors, STAT3 is constitutively phosphorylated (p) on serine 727 but not on tyrosine residues in CLL. These observations lead to investigation of the effect of STAT3/JAK2 inhibitor Cucurbitacin-I (JSI-124) in CLL cells. We proposed that the targeting STAT3 phosphorylation may induce apoptosis in CLL cells. We studied B-cell lines (BJAB, I-83, NALM-6) and primary B-cells from patients with CLL using immunoprecipitation and found that JSI-124 suppressed serine pSTAT3 and induced apoptosis of these cells as determined by FACS analysis and membrane permeability assay. Using western blotting analysis, caspase-8 activation was observed in these cells after JSI-124 treatment. Since the activation of caspases are inhibited by members of the inhibitor of apoptosis (IAP) and Bcl-2 protein families and STAT3 has been shown to regulate their expression, we investigated anti-apoptotic members of these families. JSI-124 treatment led to downregulation of IAP protein XIAP, while Bcl-xL and Mcl-1 levels remained unchanged. In addition, JSI-124 selectively induced activation of JNK signaling pathways in CLL cells. Taken together, our results indicate that JSI-124 causes apoptosis through alterations in signal transduction pathways and could be a useful treatment in CLL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 228.
Abstract 2285: Epigenetic silencing of SOD3 contributes to the malignant phenotype in human lung cancer
Authors: Fitzgerald Matthew P. , Teoh Melissa LT , Madsen Joshua M. , Domann Frederick E.
Published: February 2011

Extracellular SOD (EcSOD), encoded by the SOD3 gene, scavenges superoxide in the extracellular space. EcSOD is highly expressed in the lung where it comprises the majority of SOD activity in airway epithelial and vascular cells. The role of EcSOD in lung cancer, if any, has not yet been thoroughly studied. It has been reported, however, that EcSOD expression is lost in the lung cancer cell line A549 and that the transcription factors SP1/SP3 that regulate expression in MRC-5 cells could neither bind the promoter nor stimulate EcSOD expression in A549 cells. We hypothesized that EcSOD loss in lung cancer could be due to epigenetic events. To test this hypothesis we analyzed the epigenotype of the SOD3 promoter in three normal cell types (airway epithelial, MRC-5, HMEC) and two EcSOD negative lung cancer cell lines (A549, H1650) as well as in normal lung versus five human lung tumors. We also re-expressed EcSOD in the A549 cell line to assess possible changes in malignant phenotype. We now report that EcSOD expression is highly correlated with SOD3 promoter methylation. In the normal airway epithelial and MRC-5 lung cells that express EcSOD, the promoter has an overall cytosine methylation of <1% while in the non-expressing lung cancer cells and HMEC cells there was >86% methylation at the 18 CpG sites examined. Treatment of cells with 5-aza-dC induced SOD3 mRNA, indicating that DNA methylation was causal, at least in part, to the gene silencing. Moreover, A549 and H1650 cells displayed a ∼25-fold reduction in chromatin accessibility of the SOD3 promoter compared to MRC-5 cells. In the five lung tumors, EcSOD mRNA expression was significantly decreased and the SOD3 promoter showed a significantly more methylated state in the tumors compared to normal lung tissue. Re-expression of EcSOD attenuated the malignant phenotype of A549 cells by increasing doubling time by 25 hours, decreasing matrigel invasion and clonogenic capacity by 65% and 70% respectively. Taken together, our findings indicate that loss of EcSOD expression in human lung cancer is highly correlated with aberrant SOD3 promoter methylation and a repressive chromatin structure, and that re-acquisition of EcSOD attenuates the malignant phenotype of lung cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2285.
Abstract 2286: SPARC stimulates neuronal differentiation of medulloblastoma cells via the IL-6/STAT3 pathway
Authors: Bhoopathi Praveen , Chetty Chandramu , Gujrati Meena , Dinh Dzung H. , Rao Jasti S. , Lakka Sajani S.
Published: February 2011

Secreted Protein Acidic and Rich in Cysteine (SPARC), a 37-kDa glycoprotein, participates in the regulation of morphogenesis and cellular differentiation through its modulation of cell-matrix interactions. We have reported previously that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we show that the ectopic expression of SPARC induces neuronal differentiation in medulloblastoma cells. Adenovirus-mediated expression of SPARC cDNA (Ad-DsRed-SP) elevated the expression of neuronal markers NeuN, Nestin and MAP-2 in medulloblastoma cells. SPARC expression decreased STAT3 phosphorylation and constitutive expression of STAT3 reversed SPARC-mediated neuronal differentiation. Moreover, our results show that SPARC expression not only inhibited interleukin (IL)-6 but also attenuated IL-6-mediated STAT3 phosphorylation in medulloblastoma cells. Taken together, our results suggest that SPARC induced neuronal differentiation of medulloblastoma tumor cells through its inhibitory effect on IL-6-mediated STAT3 signaling. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed increased staining for the neuronal markers NeuN, Nestin and MAP-2. Overall, our results demonstrate that SPARC promotes neuronal differentiation and inhibits tumor growth, thereby further supporting the potential use of SPARC as a therapeutic target for medulloblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2286.
Abstract 2293: ZEB1 and ZEB2 promote EMT and invasion in esophageal squamous cell carcinoma
Authors: Naganuma Seiji , Ohashi Shinya , Kimura Sotai , Natsuizaka Mitsuteru , Gimotty Phyllis , Klein-Szanto Andres J. , Itoh Hiroshi , Nakagawa Hiroshi
Published: February 2011

Introduction: Epithelial-mesenchymal transition (EMT) promotes tumor invasion and metastasis. Amongst transcription factors essential in EMT, zinc finger E-box binding proteins ZEB1 and ZEB2 (ZEB) are upregulated in several tumor types due to loss of the miR-200 family. However, the role of ZEB in esophageal squamous cell carcinoma (ESCC) remains to be elucidated. Methods: ESCC cell lines were grown. Immortalized human esophageal cells EPC2-hTERT were retrovirally co-transduced with EGFR and mutant p53 (p53R175H), early genetic lesions common in ESCC, leading to transformation as described previously (Genes Dev. 2007; 21:2788). Short hairpin RNA was expressed by lentivirus to knockdown ZEB1 and ZEB2. Epithelial cells were grown in organotypic 3-D culture, a form of tissue engineering. Laser capture microdissection (LCM) was performed to isolate RNA for gene expression profiling in the invasive cells in 3-D culture as well as microRNA determination in archived paraffin embedded primary ESCC tissue samples. Real-time RT-PCR, Western blotting and immunohistochemistry were done to determine gene expression. Senescence-associated β-galactosidase activity was determined. Results: In primary ESCC, ZEB expression was found upregulated in tumor cells at the invasive front with increased Vimentin and decreased E-cadherin expressions. Moreover, LCM documented a significant downregulation of the miR-200 family in the invasive tumor cells consistent with EMT. ZEB upregulation and concomitant loss of the miR-200 family were also found in two out of 11 ESCC cell lines, exhibiting EMT corroborated by spindle-shaped cell morphology as well as an E-cadherin to N-cadherin class switch. EGFR overexpression and p53 mutation in EPC2-hTERT cells resulted in ZEB upregulation and a significantly augmented susceptibility to EMT, where robust induction of ZEB and suppression of the miR-200 family were observed upon TGF-β treatment. In 3-D culture, the cells co-transduced with EGFR and mutant p53 markedly invaded into the underlying stroma compartment. The invasive cells had a gene signature compatible with EMT along with ZEB upregulation. Finally, knockdown of either ZEB1 or ZEB2 not only prevented TGF-β from inducing EMT, but also TGF-β-mediated massive senescence in EPC2-hTERT cells transformed by EGFR and mutant p53. Conclusions: Our data indicate that ZEB1 and ZEB2 are negatively regulated by the miR-200 family and induced upon invasion in ESCC. ZEB may regulate senescence and EMT in concert with TGF-β signaling, thus contributing to esophageal carcinogenesis and tumor progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2293.
Abstract 23: Sanguinarine suppresses survivin expression and inhibits the growth of prostate cancer cells
Authors: Sun Meng , Chun Jae Yeon , Lou Wei , Gao Allen C.
Published: February 2011

Background: Prostate cancer is a frequently occurring disease and is the second leading cause of cancer related deaths of men in the United States. Current treatments have proved inadequate in curing or controlling prostate cancer and a search for agents for the management of this disease is urgently needed. Survivin plays an important role in both progression of castration-resistant prostate cancer and resistance to chemotherapy. Altered expression of survivin in prostate cancer cells is associated with cancer progression, drug/radiation resistance, poor prognosis and short patient survival. Methods: We performed a cell based rapid screen of the Prestwick Chemical Library consisting of 1120 FDA-approved compounds with known safety and bioavailability in humans to identify potential inhibitors of survivin and anti-cancer agents for prostate cancer. Results: Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified as a novel inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells. We found that sanguinarine inhibits survivin protein expression through protein degradation via the ubiquitin-proteasome system. Sanguinarine induces apoptosis and inhibits tumor formation and growth of human prostate cancer cells. Administration of sanguinarine, beginning 3 days post ectopic implantation of DU145 human prostate cancer cells, reduces both tumor weight and volume by more than 70% compared to the control treated mice with no apparent toxicity. In addition, sanguinarine sensitized prostate cancer cells to paclitaxel-mediated growth inhibition and apoptosis, offering a potential therapeutic strategy for overcoming taxol resistance. Conclusions: These results suggest that sanguinarine may be developed as an agent either alone or in combination with taxol for treatment of castration-resistant prostate cancer overexpressing survivin.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 23.
Abstract 230: LMP1 function in NK/T-cell lymphoma
Authors: Nuri Kim
Published: February 2011

BACKGROUND:

Because NK/T-cell lymphoma was closely associated with EBV LMP1 (latent membrane protein-1) which can directly activate NF-κB-mediated oncoproteins, therefore, in this study determined whether bortezomib (proteasome inhibitor) alone or combined with irradiation affect cytotoxicity and related mechanism in NK/T-cell lymphoma and malignant lymphoid cell lines. Beside, recently LMP1 has been introduced ambivalent function, because it's also up-regulation of Fas signaling. However, lack of information when these are activated by various stimulations. Therefore, in this study, we showed that bortezomib can enhance up-regulation of LMP1 as pro-apoptotic protein in EBV LMP1 expressed cell lines.

MATERIAL & METHOD:

Human NK/T-cell lymphoma cells (Hank-1), NK-cell lymphoma (NK-92), T-cell lymphoma cells (Jurkat), and Burkitt lymphoma cells (Raji and Jijoye) were used. Bortezomib and irradiation were treated alone or combined, and tumor cell growth and apoptosis were measured by MTT assay and FACS respectively. Apoptosis and its associated protein expression levels were measured by Western blot assay. Interaction of LMP1 and Fas were evaluated by immuno-precipitation and protein ligation assay.

RESULTS:

Cell death by combining of bortezomib and irradiation synergistically inducing in LMP1 expressed Hank-1 than no LMP1 expressed NK-92 cells. To understand the mechanism of bortezomib regulated LMP1 and Fas, we examined that activated LMP1 by bortezomib enhanced effect of irradiation inducing cytotoxicity. Combined treatment of bortezomib and irradiation up-regulated LMP1 and Fas which composed binding complex, activating Fas-related molecules. In this regard, LMP1 may have paradoxal behavior. These results provided that combination of bortezomib and irradiation enhanced LMP1 as pro-apoptotic function, leading to degradation of NF-κB and resulted caspase activation in EBV LMP1 expressed cancer cell lines.

CONCLUSION:

LMP1 is able to inducing NF-κB in normal condition as oncoprotein, however bortezomib could up-regulate LMP1, its binding with Fas in EBV LMP1 expressed lymphoid cancer cell lines. Therefore, combination of bortezomib and irradiation is suggested as potential therapy not only in NK/T-cell lymphoma but also LMP1 expressed lymphoma and tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 230.
Abstract 2300: Overexpression of Snail induces epithelial-mesenchymal transition and cancer stem cell-like phenotypes in CRC cells
Authors: Fan Fan , Evans Kurt W. , Mani Sendurai A. , Zhou Yunfei , Samuel Shaija , Lu Jia , Xia Ling , Sceusi Eric , Tozzi Federico , Ye Xiang-Cang , Ellis Lee M.
Published: February 2011

Background: Epithelial-to-mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT has been shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcriptional factor that mediates EMT in a number of tumor types. Its aberrant expression has been found in various cancers including colorectal cancer (CRC). The role of Snail in mediating EMT and CSC function in CRC has not yet been fully elucidated.

Methods: The human CRC cell lines HT29 and KM12L4 were transduced with a retrovirus containing the pBabe-puro-Snail expression construct or the empty vector. The Snail expressing cells and controls were selected with the medium containing 2µg/ml puromycin. Changes in cell proliferation and chemo-sensitivity were determined by MTT assay. Migration and invasion were determined using in vitro wound healing assay and modified Boyden Chamber assays, respectively. EMT makers (E-cadherin, vimentin) and the putative CSC marker CD133 was analyzed by Western blotting. ALDH1 enzyme activity was analyzed by the Aldefluor assay.

Results: Overexpression of Snail in CRC cells induced an EMT phenotype with elevated expression of vimentin and reduced expression of E-cadherin. Snail-induced EMT did not alter cell proliferation but showed enhanced cell motility, in which Snail-expressing cells were 5- to 6-fold more migratory than the control cells. Snail also promoted the CSC-like phenotype demonstrated by increased expression of the CSC markers CD133. The aldefluor positive cell population in the Snail-expressing cells was increased ∼4 fold. Moreover, the Snail-expressing CRC cells showed increased chemoresistance to 5FU by 50%.

Conclusion. High level of Snail expression confers the EMT and CSC phenotype in CRC cells, which enhanced cancer cell invasion and chemoresistance. Our data suggest that Snail may be a potential therapeutic target in metastatic colorectal cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2300.
Abstract 2304: Stem features associated with EMT are driven by TGFβ1 in liver-metastatic cells
Authors: Zubeldia Idoia G. , Gil-Puig Carmen , Calvo Alfonso , Larzabal Leyre , Agliano Alice , Dotor Javier , Vidal-Vanaclocha Fernando , Lecanda Jon
Published: February 2011

The liver is a major target organ for metastasis. Development of metastases requires an early release of tumour-initiating-cells from the primary tumour and adhesion of metastatic-cells into a permissive niche. We hypothesized that Epithelial-Mesenchymal Transition (EMT) underlies not only the early events, but also the promotion of the most advanced stages of a successful dissemination process to the liver. It is believed that TGFβ1 might allow tumour-initiating-cells to acquire cancer stem cell properties via EMT as a mechanism that modulates the microenvironment contributing to primary and also metastatic tumor progression. However, the mechanism of EMT induction by TGFβ1 and its involvement in liver metastases remain largely unknown. Aiming to recapitulate the hepatic metastases, we established a murine model of highly metastatic colon carcinoma cells in the context of TGFβ1-signaling. To this end, luciferase-expressing cells (MC-38luc) were pre-treated with TGFβ1 (100 pM, 48 h), before intrasplenic injection of 106 cells in C57BL/6J mice. Larger primary tumours were consistently observed and liver metastasis was achieved more rapidly after injection of MC-38luc+TGFβ1 cells, than after injection of untreated MC-38luc cells. Histological examination of liver micrometastasis also revealed slightly increased staining of Ki67, α-SMA and F4/80 markers in MC-38luc+TGFβ1 tumours. Splenic co-injection of these liver-metastatic-stem cells with non transformed hepatocytes (AML-12 cells) accelerated the incidence of liver metastasis, indicating the supportive role of the normal hepatic parenchyma. Intraperitoneal injection with a novel TGFβ1-inhibitory peptide P17 (obtained from a peptide library binding to TGFβ1) resulted in reduced liver metastasis and decrease of both CECs and CEPs. We isolated liver-metastatic cells that showed higher cellular proliferation as compared to the parental counterparts. These cells posses increased tumorogenicity in vivo, a complete lack of E-cadherin expression, and increased TGFβ1/EMT signalling properties that all relate to stem cell features. Our results provide an insight into the molecular events leading to TGFβ1-mediated malignant progression and indicate that targeting TGFβ1/EMT might represent an optimal therapeutic strategy by impairing tumor growth, stemness and further liver metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2304.
Abstract 2307: Cellular expression of E-cadherin but not its plasma membrane localization is regulated by the PTEN/PI 3 Kinase pathway via transcription factors twist and snail
Authors: Kuzel Paul F. , Nguyen Andrew , Garcia Maria Elizabeth , Hao Li , Persad Sujata
Published: February 2011

Introduction: 90% of carcinoma-related deaths in adults and children are associated with cancer metastasis. Metastasis involves phenotypical changes in cancer cells within the primary tumor. To acquire motility and invasiveness, carcinoma cells must shed many epithelial phenotypes, detach from neighboring cells and undergo a series of drastic alterations commonly referred to as the “Epithelial-Mesenchymal Transition (EMT)”. In normal epithelium, E-cadherin expression provides high-affinity anchoring to neighboring cells, serving as a tumor suppressor mechanism. With malignant transformation, N-cadherin is upregulated.

Results: Using a panel of cell lines representing the various stages of melanoma progression, [primary melanocytes (HEMA-LP); radial (WM 35); vertical (WM793) and metastatic (A2058) growth phases], we have confirmed a switch in cadherin phenotype from E- to N-cadherin. The greatest expression of E-cadherin is evident in the HEMA-LP cell line, with null expression in A2058. N-cadherin is expressed abundantly in WM793 and A2058, with almost null expression in Hema-LP. Interestingly, there is also a loss of expression of the tumor suppressor PTEN in WM793 and A2058. Transfection of wild type PTEN in A2058 cells resulted in re-expression of E-cadherin and a drop in N-cadherin levels. We also determined that the PTEN-modulated switch in cadherin profile is regulated at the transcriptional level, and involves the transcription factors Twist and Snail. Cellular expression of Twist and Snail was greatest in A2058 cells and lowest in HEMA-LP cells. Transfection of PTEN into A2058 cells caused a decrease in expression of Twist and Snail. Further, targeted knockout of Twist with shRNA in A2058 cells resulted in suppression of N-cadherin expression and the appearance of E-cadherin, while transfection of a Twist over-expression vector in HEMA-LP resulted in suppression of E-cadherin expression and the appearance of N-cadherin. Surprisingly, the invasive capacity of the A2058 cells was only reduced by 40% in the presence of PTEN. Immunofluorecent analysis of E-cadherin expression in A2058 cells indicated that this may be due to the fact that the re-expressed E-cadherin does not become targeted and localized to the plasma membrane.

Conclusion: We define a pathway that regulates cellular E- & N-cadherin expression modulated at the transcriptional level by the PTEN/PI-3 Kinase pathway and involves the transcription factors twist and snail. However, the targeted localization of E-cadherin to the plasma membrane is not regulated by this pathway but by other mechanism(s) that have not yet been identified.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2307.
Abstract 2318: Induction of Rac1b by MMP-3 causes lung tumors by activating EMT
Authors: Stallings-Mann Melody L. , Zhang Ying , Miller Erin , Radisky Derek
Published: February 2011

During development, epithelial cells acquire mesenchymal characteristics through a process known as epithelial-mesenchymal transition (EMT). Substantial evidence now exists that EMT-related processes play a major role in tumor progression. EMT is a focus of interest in lung cancer as loss of E-cadherin expression confers increased resistance to several types of chemotherapy. In previous studies, we found that matrix metalloproteinase-3 (MMP3) induced EMT in mouse mammary epithelial cells through induction of Rac1b, an alternatively spliced isoform of Rac1. We now show that exposure to MMPs stimulates Rac1b and induction of EMT markers in human lung cancer cell lines, and that knockdown of Rac1b blocks MMP induced EMT. We also show that Rac1b expression levels in human lung tumor biopsies is predictive of patient outcome, and that Rac1b and MMP expression levels are correlated in these samples, suggesting a direct relationship. We also describe the generation of new transgenic mouse models for which MMP3 or Rac1b is inducibly expressed in lung epithelial cells; activation of kRAS in these mice reveals that expression of MMP3 or Rac1b promotes increased tumor number, size, and progression to adenocarcinoma as well as increased expression of EMT markers vimentin, sm-actin, col1a1, and Snail. We also show that expression of Rac1b in lung epithelial cells leads to spontaneous adenocarcinoma formation. These results demonstrate a direct linkage between induction of EMT and tumor progression, implicate Rac1b as a key mediator of these processes, and demonstrate that MMPs can directly stimulate all of these events.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2318.
Abstract 2325: Thrombospondin 1 inhibits angiogenesis in liver metastases but not lung metastases due to differential cleavage activity of ADAMTS1
Authors: Karl Daniel L. , Lee Yoon Jin , Koch Moritz , Torres-Collado Antoni X. , Fernando Namali T. , Rothrock Courtney , Kuruppu Darshini , Ryeom Sandra , Iruela-Arispe M. Luisa , Yoon Sam S.
Published: February 2011

Background: Metastases to any organ site require angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors including thrombospondin 1 (TSP1). The principal anti-angiogenic activity of TSP1 resides in a domain containing 3 TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1.

Methods and Results: Wild-type B16F10 melanoma cells formed increased liver metastases in Tsp1-null mice compared to controls but equivalent lung metastases in Tsp1-null mice and controls. Stable cell lines of B16F10, CT26 colon carcinoma, and RenCa renal carcinoma overexpressing full-length TSP1 or 3TSR were generated. Metastases from CT26 and RenCa cell lines over-expressing TSP1 were reduced in liver but not in lung. To determine why TSP1 blocked metastatic tumor growth in the liver but not the lung, we examined cleavage of recombinant TSP1 protein by liver and lung lysates and found that liver lysate cleaved TSP1 into its 3TSR domain much more efficiently that lung lysate. TSP1-mediated inhibition of CT26 lung metastases was restored when only the 3TSR domain was over-expressed rather than full-length TSP1. Liver lysate cleavage activity was significantly abrogated when ADAMTS1 activity was (1) neutralized using ADAMTS-1 antibody, (2) knocked down by lentiviral shRNA, or (3) absent in Adamts1-null liver lysate. Livers metastases generated from B16F10 cells over-expressing TSP1 in Adamts1-haploinsufficient mice demonstrated accelerated growth compared to growth in wild-type mice.

Conclusions: TSP1 inhibits the growth of liver metastases but not lung metastases secondary to differential cleavage of TSP1 into anti-angiogenic fragments by ADAMTS1. This study emphasizes the importance of host organ environment in the regulation of tumor angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2325.
Abstract 2326: Loss of prostate-specific antigen slows growth and alters the extracellular proteome of LNCaP human prostate cancer cells
Authors: Williams Simon A. , Jelinek Christine A. , Cotter Robert J. , Isaacs John T. , Denmeade Samuel R.
Published: February 2011

Prostate-specific antigen (PSA) is the most extensively used biomarker in cancer. There continues to be intense debate regarding the use of PSA in screening for prostate cancer (PCa) and how it may be used for optimal benefit. The role PSA plays in reproductive biology and PCa has been an area of ongoing interest. As a serine protease with novel substrate preferences, PSA continues to be secreted at high levels (mg/mL) even by the majority of advanced prostatic carcinomas. This protease has been shown to affect the growth or viability of a variety of cell types, including epithelial cells, osteoclasts, T-cells, and human PCa cell lines.

In this study, we stably reduced endogenous PSA gene expression using lentiviral short hairpin loop RNA (shRNA) technology in the LNCaP human PCa cell line. We then investigated any resulting changes to LNCaP growth rates in vitro and ex vivo. Acting on the hypothesis that any PSA-induced changes to LNCaP behavior are the result of the enzyme's extracellular activity, we used a high-throughput shot-gun mass spectrometry-based (MS) approach to analyze the in vitro extracellular LNCaP proteome in control and PSA-targeted shRNA populations.

Having demonstrated that our shRNA strategy targeted to PSA caused as much as a 90% reduction in PSA protein production compared to control cells, we next showed that in vitro doubling times are slowed from 2 days to 11 days. Subcutaneous tumors displayed an almost 50% reduction in take rate, with the final weights of those remaining only reaching 10% of controls after 8 weeks of growth.

Serum-free media conditioned for 24 hours by control or PSA-targeted LNCaP populations were then subjected to global semi-quantitative proteomics analysis using the high resolution LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA). In one 200 minute LTQ-Orbitrap analysis, in conjunction with the Mascot Search algorithm (Matrix Science, Boston, MA), we were able to identify 1784 proteins in the LNCaP conditioned media. Of these, 686 were specific to the control populations, 527 were specific to the reduced PSA populations, and 571 were common to both.

These results demonstrate the utility of high-throughput proteomic strategies as an unbiased approach for the identification of candidate mechanisms behind novel observations in cancer biology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2326.
Abstract 2328: Hyaluronan synthase expression in mammary tumor cells modulates tumor growth, angiogenesis and metastasis
Authors: Varney Michelle L. , Singh Rakesh
Published: February 2011

Breast cancer is the second most common cause of cancer death in women in the United States after lung cancer and is responsible for more than 40,000 deaths each year. At the time of diagnosis, the majority of breast cancer patients have developed regional lymph node or distant metastases. Many challenges exist in the current management of metastatic breast cancer as there are fewer recognized therapeutic strategies. Therefore, a better understanding of the molecular events in the metastatic process is critical. Several reports have described correlation of hyaluronan (HA) with initiation and progression of various types of epithelial cancers. The HA synthase (HAS) isoforms encode the enzymes that produce and deposit HA while the hyaluronidase (HYAL) genes code for enzymes that degrade HA and their expression is dysregulated in various tumors as a result of transcriptional and epigenetic changes that accompany progression. In this report, we examined the expression of HAS1, HAS2 and HAS3, HYAL1 and HYAL2 in mammary tumor cell with different metastatic potential (4T1, highly metastatic; Cl66, moderately metastatic; Cl66M2, low metastatic). We observed increased expression of HAS1, HAS2 and HAS3 as well as HAYL 1 and HAYL2 in aggressive mammary tumor cells. Next we stably knocked-down HAS2 and HAS3 expression in 4T1 cells using small hairpin mRNA (sh-RNA) vectors and analyzed cell proliferation, migration, tumor growth and metastasis. We observed inhibition of in vitro cell proliferation and migration in 4T1 cells knocked-down for HAS2 (4T1-HAS2sh) and HAS3 (4T1-HAS3sh) as compared to cells transfected with vector control (4T1-controlsh). Furthermore, we observed inhibition of in vivo tumor growth and spontaneous lymph node and lung metastases in animals implanted with 4T1-HAS2sh cells as compared to 4T1-controlsh. In addition, we observed inhibition of tumor cell proliferation and neovascularization, and increased apoptosis in 4T1-shHAS2 tumors as compared to 4T1-controlsh tumors. Together, these data demonstrate an important role of HAS2 in mammary tumor growth, angiogenesis, invasion, and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2328.