bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 233: OTX2 copy number gain is a driver event in Shh-independent medulloblastomas
Authors: Wortham Matthew , Adamson David C. , Shi Qun , Northcott Paul A. , Di Chunhui , Duncan Christopher G. , Li Jianjun , McLendon Roger E. , Bigner Darell D. , Taylor Michael D. , Yan Hai
Published: February 2011

Medulloblastoma is the most common malignant brain tumor in children. With the exception of the Sonic Hedgehog (Shh) and Wnt pathways, the molecular events contributing to medulloblastoma tumorigenesis are poorly understood. In this study, we performed DNA copy number analysis upon a large cohort (n=201) of primary medulloblastomas to identify recurrent copy number events of these tumors. The most commonly gained region of the medulloblastoma genome (gained in 21% of tumors) contains the single gene OTX2, which encodes a developmental transcription factor involved in regionalization of the neural tube and specification of various CNS lineages. We found that OTX2 is also commonly overexpressed (74%, n=103) in these tumors. Molecular classification of medulloblastomas identified four distinct subtypes based on gene expression signature. OTX2 overexpression and copy number gain was limited to tumors not exhibiting a signature of Shh pathway activation. We found that OTX2 is necessary for intracranial xenograft formation by medulloblastoma cells and that its expression promotes xenograft tumor formation by immortalized primary cells. Finally, we determined that OTX2 regulates the medulloblastoma oncogene MYC. As a transcription factor, targeting OTX2 function with small molecules presents a challenge. However, retinoids can reduce OTX2 expression and inhibit medulloblastoma growth in vitro, and we have demonstrated that OTX2 silencing is required for this effect. In sum, these studies identify OTX2 as a common but targetable oncogene in Shh-independent medulloblastomas.

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 233.
Abstract 2330: MMP-2 regulates Rac1- and Cdc42-mediated cell migration via integrin αVβ3/FAK in U87 and U251 glioblastoma cell lines
Authors: Chetty Chandramu , Lakka Sajani S. , Bhoopathi Praveen , Fassett Daniel , Klopfenstein Jeffrey D. , Dinh Dzung H. , Gujrati Meena , Rao Jasti S.
Published: February 2011

Gliomas, the most common form of brain tumors, are characterized by a capacity to invade through normal brain tissue. This infiltrative character is the hallmark of poor prognosis. A greater understanding of the molecular determinants that drive cell motility may lead to improved therapy. The molecules of the extracellular matrix are involved in organization of the cytoskeleton and activation of intracellular signaling that is required for the regulation of cell adhesion and migration. Here, we show that the downregulation of matrix metalloproteinase-2 (MMP-2; gelatinase A) using an adenoviral vector expressing siRNA (Ad-MMP-2-Si) inhibited cell migration in U87 and U251 glioblastoma cell lines as determined by spheroid migration and wound-healing assays. Transcriptional inactivation of MMP-2 altered interaction of MMP-2 and the integrin-αVβ3 as determined by immunoprecipitation and co-localization studies. In addition, downregulation of MMP-2 inhibited the complex formation of FAK, integrin-αVβ3 and 14-3-3ζ, which in turn decreased the activation of GTP-bound forms of Rac1 and Cdc42. Transfection of the cells with constitutively active Rac1 and Cdc42 reversed Ad-MMP-2-Si-blocked cell migration and gelatin degrading. Further, administration of recombinant human MMP-2 reversed MMP-2 siRNA-inhibited GTP-bound forms of Rac1 and Cdc42. Additionally, functionally blocking integrin-αVβ3 antibody inhibited GTP-bound forms of Rac1 and Cdc42, thereby revealing that the interaction of MMP-2 and integrin-αVβ3 plays a critical role in cell migration. In summary, our data demonstrate that the inhibition of MMP-2 inhibits integrin-αVβ3/FAK-mediated migration by reducing the GTP-bound forms of Rac1 and Cdc42 in U87 and U251 glioblastoma cell lines.

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 2330.
Abstract 2331: One of the first steps of metastasis is remodeling of the extracellular matrix microenvironment
Authors: Kenny Hilary A. , Leonhardt Payton , Ladanyi Andras , Gwin Katja , Lengyel Ernst
Published: February 2011

During metastasis tumor cells initiate processes that lead to the loss of normal tissue architecture and formation of the tumor microenvironment. The main site of metastasis in ovarian cancer (OvCa) is the omentum. Little is known about the initial changes in extra-cellular matrix (ECM) during early OvCa metastasis. A 3-dimensional (D) organotypic model of the omentum was used to investigate the remodeling of the ECMs during the initial steps of OvCa metastasis. Vitronectin and fibronectin increased when OvCa cells (SKOV3ip1 and Hey A8) were added to the 3D omental model, while collagen type I decreased. Vitronectin was produced by the cancer cells and stroma, while fibronectin was produced solely by cancer cells. Collagen type I decreases in stroma and increases in OvCa when co-cultured. Vitronectin, fibronectin and collagen type I are produced by primary human mesothelial cells and fibroblasts and are present in ascites. Two immunohistochemical studies with human tumors confirmed the above findings. First during early OvCa metastasis in humans, fibronectin is present in the stroma, while vitronectin and collagen type I are present in both cancer cells and the stroma. Second, ECM localization in 20 sets of primary and omental metastasis tumors from the same patients was performed. Fibronectin was absent in the primary tumor and present in the metastatic tumor stroma, while vitronectin was produced in cancer cells and stroma in primary and metastatic tumors. Additionally, a Reverse-Phase Protein Array was performed on primary versus omental metastasis tissue from 20 postmenopausal patients with FIGO Stage III/IV, high grade papillary-serous OvCa. Fibronectin was significantly increased, while vitronectin was significantly decreased in the omental metastasis vs. primary tumor, respectively. Additional experiments revealed changes in the tumor cell cytoskeleton in conjunction with increased expression of integrins (α5β1-and αvβ3-integrin) and proteases (uPA, uPAR, MMP-2 and MMP-9). Integrins and proteases were transiently knocked down using sequence-specific siRNA. Both α5β1-integrin and MMP-2, but not αvβ3-integrin and MMP-9, were found to be necessary for the remodeling of fibronectin. Taken together, these findings reveal the initial transformation of ECM in the normal omental architecture during early OvCa 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 2331.
Abstract 2335: Role of heparanase in colitis associated cancer
Authors: Elkin Michael , Lerner Immanuel , Sherman Yoav , Hermano Esther , Vlodavsky Israel
Published: February 2011

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Here we report that heparanase enzyme acts as an important mediator of colitis-associated tumorigenesis. Heparanase is an only known mammalian enzyme that cleaves heparan sulfate, the major polysaccharide of the extracellular matrix, and plays multiple roles in inflammation and cancer progression. Applying histological specimens from UC patients and a mouse model of dextran sulfate sodium (DSS)-induced colitis, we found that heparanase is constantly overexpressed and activated during the course of the disease, both in the active and inactive phases of inflammation. Employing heparanase-overexpressing transgenic mice in the model of colitis-associated cancer, induced by carcinogen azoxymethane followed by repeated DSS administration, we demonstrated that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors, enabling faster tumor take, angiogenic switch and enhanced tumor progression. Notably, DSS-induced colitis alone (without azoxymethane pretreatment) lead to formation of colonic tumors in heparanase-transgenic, but not wild type mice, positioning heparanase as important physiological determinant in inflammation-driven colon carcinoma, replacing the need for carcinogen. Investigating molecular mechanisms underlying heparanase induction in colitis, we found that macrophage-derived cytokines (i.e., TNFalfa) are responsible for continuous overexpression of heparanase by chronically-inflamed colonic epithelium. Moreover, our results suggest the occurrence of heparanase-driven vicious cycle that power colitis and associated tumorigenesis: heparanase activity in inflamed colon, acting synergistically with the local cytokine milieu, stimulates macrophage activation, and the activated macrophages secrete TNFalfa which stimulate further production of heparanase by colonic epithelium. In addition, activated macrophages secrete cathepsin L - a cysteine protease responsible for proteolytic activation of latent heparanase enzyme. Altogether, our results identify heparanase as a key factor in pathogenesis of colitis-associated cancer and attest macrophages as both heparanase regulators and cellular target for heparanase action.

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 2335.
Abstract 2337: Effect of embryonic stem (ES) cell-conditioned microenvironment on the reprogramming of human cancer
Authors: Kim Myoung Ok , Cho Yong-Yeon , Kim Sung-Hyun , Jeong Chul-Ho , Lee Kun-Yeong , Jeon Young-Jin , Bode Ann M. , Dong Zigang
Published: February 2011

During embryonic development, the extracellular matrix (ECM) plays a critical role in the regulation of stem cells to different lineages, cell migration and proliferation. A recently developed 3-dimensional (3D) ES-conditioned matrigel matrix culture of metastatic melanoma cells induces reprogramming and differentiation. However, although the ES-cell conditioned microenvironment induces melanoma cell reprogramming to suppress invasive behavior, the mechanisms have not yet been clearly understood. To investigate the effect of the cancer cell microenvironment, we hypothesized that 1) the microenvironment might induce nuclear reprogramming; 2) microenvironment-induced reprogramming might affect cell proliferation; 3) microenvironment-induced reprogramming might induce differentiation-mediated cellular senescence; and 4) microenvironment-induced reprogramming causes a loss of anchorage-independent cell growth, which is a hallmark of cancer cell properties. Here, we found that the ES-conditioned 3D-microenvironment induced a dramatic change in the morphology of A431 human skin epidermoid carcinoma cells and SK-MEL-28 human skin malignant melanoma cells. In the anchorage-independent cell growth assay, we found that A431 and SK-MEL-28 cells grown in the 3D ES-conditioned microenvironment exhibited dramatically decreased colony growth in soft agar. Real time PCR analysis indicated that ES-conditioned microenvironment suppressed endogenous Oct4 and Nanog gene expression in A431 and SK-MEL-28 cells and inhibited c-jun and c-fos promoter activities, which are components of the AP-1 transcriptional factor complex. Very importantly, we found that the ES cell-conditioned microenvironment inhibited telomerase activity, resulting in the induction of cellular senescence in human skin melanoma cells. We found that ES-conditioned medium contained a greater amount of Gremlin, a natural inhibitor of TGF-β signaling. Gremlin treatment of A431 and SK-MEL-28 cells induced morphological changes as observed in the 3D ES-conditioned matrigel matrix. Our results demonstrated that the ES-cell conditioned microenvironment inhibited proliferation and induced cellular senescence of A431 and SK-Mel-28 cells through the inhibition of TGF-β signaling by Gremlin. Furthermore, our data strongly suggests that modulation of TGF-β signaling in the cancer microenvironment is a potential strategy to modulate cancer cell behaviors and properties.

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 2337.
Abstract 2338: Molecular mechanisms underlying cellular transformation by Tumor protein D52 overexpression
Authors: Della-Franca Austin , Catchpoole Daniel R. , Bright Robert K. , deFazio Anna , Byrne Jennifer A.
Published: February 2011

Background: Tumor protein D52 (D52) overexpression is characteristic of both adult and childhood cancers and has been associated with reduced overall survival in breast cancer patients and an increase in proliferation and metastatic capacity of transfected cell lines. However, little is known about the mechanism(s) by which D52 overexpression contributes to cellular transformation or tumour progression.

Aims: We aimed to identify genes which are differentially expressed in response to D52 expression in 3T3 fibroblasts, and to use these gene expression changes to predict cellular phenotypes which are altered by D52 expression.

Methods: Gene expression profiling was performed using Affymetrix expression microarray analysis. Up- and down-regulated gene lists were analysed using GOSTAT, Metacore and literature searches. Differential expression of candidate proteins were validated using Western blot analysis of intracellular protein extracts or precipitates from conditioned media. Migration and invasion assays were performed using Boyden chambers.

Results: D52 expression was associated with over 300 genes being differentially expressed at least two-fold relative to vector controls. A number of overexpressed genes were found to be normally involved in the inflammatory response. We focused on validating candidates such as COX-2, MMP-2 and Osteopontin as being overexpressed in D52-transfected cells relative to vector controls. We also confirmed an upregulation of phospho-AKT in D52 expressing cells, as reported by others. GOSTAT analysis revealed that gene ontology terms related to the extracellular matrix (ECM) were most over-represented amongst differentially expressed genes. The implied resultant ECM re-modelling was examined through migration and invasion assays, in which D52 expressing cells showed a significantly increased migratory and invasive capacity relative to vector control lines.

Conclusions: These results implicate D52 as a pro-inflammatory intermediate, whose expression may act to drive cancer progression via an increase in phospho-AKT, followed by the production of inflammatory molecules such as Osteopontin.

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 2338.
Abstract 234: Transcription factor LSF is a novel oncogene for hepatocellular carcinoma (HCC)
Authors: Yoo Byoung Kwon , Emdad Luni , Gredler Rachel , Chen Dong , Su Zhao-zhong , Fisher Paul B. , Sarkar Devanand
Published: February 2011

While the incidence of overall cancers is decreasing, Hepatocellular Carcinoma (HCC) is one of the few cancers the incidence of which is on an increase. The mortality rate of HCC parallels that of incidence since HCC is a highly aggressive cancer with extremely limited currently available therapeutic strategies. Understanding the molecular mechanism of hepatocarcinogenesis will help develop novel, targeted and more effective therapeutic regimen to counteract this fatal disease. Here, we demonstrate that the cellular transcriptional factor Late SV40 Factor (LSF) functions as an oncogene in HCC. The level of LSF expression is significantly increased in human HCC patients compared to matched non-neoplastic controls and normal liver and LSF expression significantly correlates with the stages and grades of HCC. Ectopic overexpression of LSF in nontumorigenic human HCC cells leads to highly aggressive, multi-organ metastatic tumors while inhibition of LSF in aggressive human HCC cells profoundly inhibits tumorigenic and metastatic properties in athymic nude mice. Affymetrix microarray studies identified novel LSF-downstream genes that are associated with cell growth, angiogenesis, invasion, metastasis, chemoresistance, and senescence. Osteopontin, a key molecule mediating tumor progression, especially metastasis, was identified as a downstream target gene of LSF. Promoter-reporter and chromatin immunoprecipitation analyses confirmed that LSF transcriptionally upregulates OPN by binding to its promoter. Knockdown of OPN gene in LSF-overexpressing HCC cells markedly inhibits LSF-mediated tumor progression including metastasis. This is the first report on the novel function of LSF as an oncogene in any cancer indication. Since LSF expression correlates with the advanced stages of the disease, it might be employed as a diagnostic/prognostic marker for HCC. The DNA binding domain of LSF might be targeted with small molecule inhibitors that might be developed as potential HCC therapeutic. Our findings thus have significant translational implications.

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 234.
Abstract 2343: Overexpression of uPAR in the murine oral squamous cell carcinoma cell line AT84
Authors: Magnussen Synnøve , Haustreis Stine , Uhlin-Hansen Lars , Winberg Jan-Olof , Svineng Gunbjorg
Published: February 2011

Cancers of the oral cavity are associated with bad prognosis and have been so for the last 20-30 years. Almost half of the patients diagnosed with oral squamous cell carcinoma (SCC) will die from the disease. When comparing oral SCC to its counterpart in skin, one of the main features that differ between the two is the ability of the oral SCC to metastasize. A prerequisite for metastasis is the capability to invade the surrounding tissue and penetrate into the nearest blood- or lymphatic vessel. This initial step in metastasis requires several extracellular matrix degrading enzymes. One such enzyme is uPA (urokinase-type plasminogen activator), involved in pericellular proteolysis, where high levels are known to be correlated to bad prognosis in several cancers. Pro-uPA binds to its extracellularly located receptor uPAR. Upon binding, pro-uPA is cleaved and activated, and ample evidence shows that binding of uPA triggers uPAR-mediated cellular signalling. Being that uPAR has no membrane spanning domain, the signal transduction has to be mediated through one or several membrane partners. uPARs role in the regulation of cell adhesion, migration and proliferation have been dedicated to its interplay with membrane partners such as integrins, chemokine receptors, EGFR, G-protein coupled receptors and caveolin. To better understand the role of uPAR in the metastasis of oral SCCs, a mouse oral SCC cell line, AT84, was stably transfected with uPAR. AT84 was initially obtained from a spontaneously originating oral SCC of the C3H mouse (Lou et al., 2003; Schultz-Hector and Haghayegh, 1993). Characterization of these cell lines with respect to adhesion, migration, invasion, and effects on gene expression and activation of other proteolytic enzymes will be presented.

Lou, E., R.M. Kellman, R. Hutchison, and E.J. Shillitoe. 2003. Clinical and pathological features of the murine AT-84 orthotopic model of oral cancer. Oral Dis. 9:305-12.

Schultz-Hector, S., and S. Haghayegh. 1993. Beta-fibroblast growth factor expression in human and murine squamous cell carcinomas and its relationship to regional endothelial cell proliferation. Cancer Res. 53:1444-9.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 2343.
Abstract 2344: Soluble MICA in cervical cancer is in a deglycosylated form: Implication for soluble MICA detection
Authors: Nanok C , Barusrux Sahapat , Leelayuwat C
Published: February 2011

Background: Major histocompatibility complex class I chain related A (MICA/PERB11.1) molecules are expressed as transmembrane proteins. They are ligands recognized by the natural killer cell receptor group (NKG)2D, an activating receptor on NK cells, gamma delta T cells and activated CD8 T cells. Up-regulated MICA expression was reported in many tumors. Shedding of MICA from tumor cells as soluble MICA (sMICA) is a mechanism for immune evasion. MICA proteins could be presented in various sizes from 43 to 75 kDa depending on degree of glycosylation. Eight potential N-glycosylation sites in a MICA molecule were reported. This study aimed to investigate the size of sMICA in sera of cervical cancer patients and women blood donors by western blot technique.

Methods: The sMICA*009 plasmid DNA was transfected to COS7 cells for recombinant sMICA protein production in culture supernatant. The recombinant sMICA protein was harvested and validated with mouse monoclonal anti-MICA antibody (WW6B7) by indirect ELISA and immunobloting. Molecular weight of deglycosylated recombinant sMICA protein treated with N-glycosidase F enzyme and sMICA in cervical cancer and healthy sera was determined.

Results: The recombinant sMICA and serum sMICA showed 62 kDa and 45 kDa in molecular weight, respectively. The molecular weight of recombinant sMICA treated with N-glycosidase F enzyme was reduced to 45 kDa which equal to serum sMICA indicating that sMICA in sera of patients and blood donor controls was in a deglycosylated form.

Conclusion: Recombinant sMICA produced in COS7 had a molecular weight of 62 kDa. In contrast, sMICA in sera was in a deglycosylated form and had a molecular weight of 45 kDa equal to in vitro deglycosylation with N-glycosidase F enzyme of produced recombinant sMICA. The mechanism and kinetic of deglycosylation of sMICA in sera await further investigation. It would be interesting to see whether this form of sMICA would affect sMICA detection in sera.

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 2344.
Abstract 2350: MicroRNA-145 suppresses cell invasion and metastasis by directly targeting mucin 1
Authors: Sachdeva Mohit , Mo Yin Yuan
Published: February 2011

MicroRNAs (miRNAs) are important gene regulators that could play a profound role in tumorigenesis. Our previous studies indicate that miR-145 is a tumor suppressor capable of inhibiting tumor cell growth both in vitro and in vivo. In this study, we show that miR-145 exerts its function in a cell-specific manner. Although miR-145 inhibits cell growth in MCF-7 and HCT-116 cells, it has no significant effect on cell growth in metastatic breast cancer cell lines. However, miR-145 significantly suppresses cell invasion in these cells, as shown by matrigel chamber assays; in contrast, the antisense oligo against miR-145 increases cell invasion. MiR-145 is also able to suppress lung metastasis in an experimental metastasis animal model. This miR-145-mediated suppression of cell invasion is in part due to the silencing of the metastasis gene mucin 1 (MUC1). Using luciferase reporters carrying the 3′-untranslated region of MUC1 combined with western blot and immunofluorescence staining, we identify MUC1 as a direct target of miR-145. Moreover, ectopic expression of MUC1 enhances cell invasion, which can be blocked by miR-145. Of interest, suppression of MUC1 by miR-145 causes a reduction of β-catenin as well as the oncogenic cadherin 11. Finally, suppression of MUC1 by RNAi mimics the miR-145 action in suppression of invasion, which is associated with downregulation of β-catenin and cadherin 11. Taken together, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor growth, but also cell 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 2350.
Abstract 2353: CREB regulates AP-2α transcription to determine the malignant phenotype of melanoma
Authors: Dobroff Andrey S. , Melnikova Vladislava O. , Villares Gabriel J. , Zigler Maya , Braeuer Russell R. , Wang Hua , Huang Li , Bar-Eli Menashe
Published: February 2011

The Activator protein-2 (AP-2) family of transcription factors consists of five different proteins which are involved in the regulation of cell proliferation, differentiation, apoptosis and carcinogenesis. Among these proteins, AP-2α has been suggested to function as a tumor suppressor in many cancers including melanoma. We have previously shown that in melanoma cell lines, acquisition of the metastatic phenotype is associated with loss of AP-2α expression and gain in expression of cAMP-responsive element (CRE)-binding protein (CREB). This inverse correlation is associated with increase of melanoma invasion and metastasis resulting in overexpression of the melanoma cell adhesion molecule MCAM/MUC18 and protease-activated receptor 1 (PAR-1). However, the molecular mechanism involved in AP-2α regulation during melanoma progression remains unknown. Analyses of AP-2α promoter for transcription factor binding sites showed two distinct CRE-like sites as well as four E2F1 sites. Herein, we propose that the inhibition of AP-2α during the transition from the Radial Growth Phase (RGP) to the Vertical Growth Phase (VGP) occurs at the transcriptional level and is regulated through binding of CREB and E2F1. Hence, AP-2α protein expression was found to be increased after utilizing small hairpin RNA (shRNA) targeting CREB in two human metastatic melanoma cell lines, A375SM and C8161. Interestingly, E2F1 expression level was found to be reduced by 50% after CREB silencing in both cell lines. We also found a 20-fold increase in the luciferase activity driven by the AP-2α promoter after silencing CREB in both cell lines. In addition, point mutations at CRE-like binding sites as well as E2F1 increased the AP-2α promoter activity by more then 5-fold. To further establish the involvement of E2F1 in AP-2α regulation, we transiently overexpressed E2F1 in a low-metastatic melanoma cell line, SB-2. The AP-2α protein level as well as the promoter activity was significantly inhibited as compared to control. Finally, using Chromatin Immunoprecipitation (ChIP) assays in both CREB silenced cell lines, we were able to confirm that CREB and E2F1 bind to the AP-2α promoter thereby inhibiting its expression. Taken together, our studies demonstrate that CREB regulates AP-2α expression, thus, contributing to the metastatic phenotype of melanoma.

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 2353.
Abstract 2356: The role of the Rac GTPases in PC-3 prostate cancer cell diapadesis
Authors: Pathak Surabhi , Chatterjee Moumita , Dubyk Cara W. , Sequeira Linda , van Golen Kenneth
Published: February 2011

The Rho GTPases are molecular switches intimately involved in cancer metastasis through orchestrating changes in the cytoskeleton leading to motility and invasion. Previously, we demonstrated that RhoC GTPase was sufficient and required for PC-3 prostate cancer invasion. Furthermore, we demonstrated that targeted down-regulation of RhoC using either a dominant-negative RhoC or by specific shRNAs led to increased and sustained activation of Rac1 GTPase. Increased levels of active Rac1 led to morphological, molecular and phenotypic changes reminiscent of epithelial to mesenchymal transition (EMT). Additionally, we demonstrated a requirement for active Rac1 GTPase in PC-3 tumor cell diapedesis across a bone marrow endothelial cell (BMEC) layer, an important step in prostate cancer skeletal metastasis. In the current study, we analyze the specific roles of Rac1, Rac3 and RhoG GTPases in tumor cell diapadesis. Use of specific siRNAs to down-regulate the individual Rac proteins demonstrated unique roles for each in promoting cell adhesion and diapedesis. We found Rac1 to be expressed at significantly higher levels than Rac3 or RhoG. However, RhoG appears to regulate PC-3 diapadesis at levels similar to Rac1. In contrast, Rac3 does not regulate diapadesis but controls PC-3 binding to substrates and BMECs. Together, these data suggest a role for coordinated activity of the Rac GTPases during diapadesis.

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 2356.
Abstract 2357: Defining the Ezrin-CLIC4 interaction in osteosarcoma
Authors: Mendoza Martin , Ren Ling , Davicioni Elai , Wai Daniel , Triche Timothy , Khanna Chand
Published: February 2011

Ezrin, a linker protein between the cytoskeleton and plasma membrane, plays an important role in the metastatic phenotype of cancers that includes osteosarcoma and rhabdomyosarcoma. To better understand the role of ezrin in metastasis, we have identified proteins that bind the N-terminus of ezrin using tandem mass spectrometry affinity chromatography. One of the mass spectrometry identified proteins is the p53, TNFα, and c-Myc regulated stress-response protein Chloride Intracellular Channel 4 (CLIC4). Under conditions of cell stress, CLIC4 has been shown to translocate to the nucleus and subsequently cause cell-cycle arrest and/or accelerate apoptosis in cancer cells including osteosarcoma. Emerging data from our lab suggests that ezrin may provide metastatic cells with an early survival advantage by helping to resist the stresses of metastasis and resultant apoptosis. Combining these two lines of reasoning, we hypothesized that ezrin prevents CLIC4 induced apoptosis during times of stress by maintaining CLIC4 out of the nucleus. Using cell free conditions we have demonstrated a direct relationship between ezrin and CLIC4 recombinant proteins and through co-immunoprecipitation in osteosarcoma cells. In addition, experiments to define the minimal binding determinants of the ezrin-CLIC4 interaction are currently underway. We have confirmed co-expression of ezrin and CLIC4 in multiple human osteosarcoma cell lines and human sarcomas using both immunohistochemistry and expression microarray. We have recently developed assays and reagents to monitor and manipulate CLIC4 nuclear localization under conditions of stress. The outcome of this work will be to define the nature and biological significance of the ezrin-CLIC4 interaction in 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 2357.
Abstract 2382: IGFBP-3 regulates angiogenesis through upregulation of TSP-1
Authors: Torng Pao-Ling , Shih Ho-Jun , Huang Su-Cheng , Lin Chin-Tarng
Published: February 2011

Insulin growth factor binding protein-3 (IGFBP-3) has been postulated to be a mediator of growth suppression signal. In our previous study, IGFBP-3 was found as an invasion suppressor gene in ovarian endometrioid carcinoma (OEC) (Oncogene 2008;27:2137-47). It was silenced through IGFBP-3 promoter methylation, which was associated with cancer progression (Mol Cancer, in press). Through microarray analysis, we further identified that IGFBP-3 expression was associated with an elevation of thrombospondin-1 (TSP-1), which is an endogenous inhibitor of angiogenesis. We established a tetracyclin-inducible IGFBP-3 expressing OEC cell line using pBIG2i plasmid (OVTW59 P4-D-I: pBIG2i-hIGFBP3). This cell line showed 4-fold increase in IGFBP-3 expression 2 hours after tetracyclin treatment, associated with an paraellel increase in TSP-1. Expression of IGFBP-3 decreases cell migration and capillary tube formation, which was reversed after anti-TSP-1 treatment. By chick embryo chorioallantoic membrane (CAM) assay, IGFBP-3 expression showed a significant decrease in capillary formation. Heterotransplantation of IGFBP-3-transfectants significantly decrease in tumor growth 5 days after IGFBP-3 induction. Such dormancy state of tumor growth persisted for about 10 days. Xenograft tumors during this period showed a decrease in microvessel density and an increase in tumor apoptosis. In conclusion, we have identified a novel association between IGFBP-3 expression and TSP-1 elevation that consequently results in a decrease in angiogenesis. Such impairment in angiogensis could be associated with the dormancy state of cancer growth and may represent a major function of the oncosupressor IGFBP-3 in ovarian cancer pathogenesis.

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 2382.
Abstract 2383: MicroRNA-519c suppresses HIF-1α expression and tumor angiogenesis
Authors: Cha Shih-Ting , Chen Pai-Sheng
Published: February 2011

Hypoxia-inducible factor (HIF)-1α is widely considered to be one of the key regulators of tumor angiogenesis. The upstream regulation is complex and involves several growth factors, cytokines and hypoxia. Herein, we identified miR-519c as a hypoxia independent regulator of HIF-1α, acting through direct binding to HIF-1α 3′UTR and leading to reduced tumor angiogenesis. Over expression of miR-519c resulted in a significant decrease of HIF-1α protein levels and reduced the tube formation of HUVEC cells and similarly, antagomir inhibition of miR-519c increased the level of HIF-1α protein and enhanced angiogenic activity, suggesting an important role of miR-519c in HIF-1α-mediated angiogenesis. In consistence with the over-expression of miR-519c in cancer patients with better prognosis, mice injected with miR-519c over-expressing cells exhibited dramatically reduced HIF-1α level, followed by suppressed tumor angiogenesis, growth, and metastasis. In addition, we found that hepatocyte growth factor (HGF), a known HIF-1α inducer, reduced miR-519c levels in an Akt-dependent pathway. This regulation is post-transcriptional and may mediate by suppressing miR-519c maturation. Taken together, our findings provide the first evidence that miR-519c is a pivotal regulator of tumor angiogenesis and, moreover, micro environmental HGF contributes to regulating miR-519c biogenesis in cancer cells.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 2383.
Abstract 2387: Characterization the role of leukocyte cell-derived chemotaxin 2 (LECT2) in angiogenesis
Authors: Chi-Kuan Chen , Chung Wan-Chin , Wu Yu-Ling , Yang Ching-Yao , Chang Jia-Ming , Kuo Ming-Lian
Published: February 2011

Leukocyte Cell-Derived Chemotaxin 2 (LECT2) protein is expressed preferentially in the liver in a constitutive manner and secreted into bloodstream. Recently studies indicate that LECT2 potentially associated with the liver injury, repair, and regeneration, but other functions are still unknown. Angiogenesis is a process of new blood vessel formation which has essential roles in development, repair, tumor growth, invasion, and metastasis. In our pervious studies, we have recently identified LECT2 as a potential tumor suppressor, correlating with decreased vascular invasion in hepatocellular carcinomas (HCC). In this study, we evaluated the function of recombinant LECT2 (rLECT2) proteins on angiogenesis by using human umbilical vein endothelial cells (HUVEC). We demonstrated a selective and significant inhibition of VEGF165-induced angiogenic activity in HUVEC by rLECT2 protein through inhibiting the proliferation, migration, and tube formation. The rLECT2 protein also suppressed VEGF165-induced angiogenesis in CAM assay and matrigel plug assay. In conclusion, we for the first time found that LECT2 played an important role in anti-angiogenesis. Moreover, the LECT2 might have broad therapeutic applications in diseases characterized by excessive angiogenesis.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 2387.
Abstract 2392: Transcriptional repression of VEGF by Znf24, a C2H2 zinc finger protein
Authors: Jia Di , Loureiro Robyn , D'Amore Patricia , Rodig Scott , Moses Marsha
Published: February 2011

Vascular endothelial growth factor (VEGF) is a key regulator during the formation of new blood vessels during angiogenesis. It functions to promote the proliferation and migration of endothelial cells under both physiological conditions such as embryogenesis and pathological conditions such as tumor growth. VEGF has been shown to play an important role in the development and metastasis of breast as well as other human cancers. To date, many trans-activating factors of VEGF have been described, whereas transcriptional repression of VEGF remains less studied. We have previously reported the identification of a SCAN-domain containing C2H2 zinc finger protein, Znf24, which functions to repress the transcription of VEGF. In this study we explore the mechanism by which Znf24 represses VEGF transcription. We have generated a series of VEGF promoter constructs bearing deletions from the 5′ end. Reporter gene assays in the breast cancer cell line MDA-MB-231 have revealed a proximal region of the VEGF promoter essential for Znf24-induced repression. To determine whether Znf24 represses VEGF transcription through direct binding to the VEGF promoter, we have performed electrophoretic mobility shift assays (EMSA) using the VEGF proximal promoter region as a probe. We have found that Znf24 protein binds to this proximal region, suggesting that VEGF is a direct downstream target of Znf24. To begin to investigate Znf24 functions in vivo, we have conducted immunohistochemistry assays using human breast cancer tissue microarrays representing different stages of disease progression and tumor sizes, as well as matched normal breast tissues. We have determined that Znf24 protein levels are significantly decreased in malignant tissues compared to normal adjacent tissues. A potential negative correlation between Znf24 protein levels and tumor sizes is also observed, suggesting that Znf24 may serve as a negative regulator of tumor growth. Our data suggest that Znf24 represses VEGF transcription through direct binding to the VEGF promoter and that it may function as a negative regulator of tumor growth by inhibiting tumor angiogenesis. (Supported by NIH P01 CA045548 and the Simeon J. Fortin Charitable Foundation)

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 2392.
Abstract 2397: SSX-2 vaccines with enhanced MHC class I binding epitopes elicit anti-prostate tumor CTL
Authors: Smith Heath A. , McNeel Douglas G.
Published: February 2011

The tumor-associated antigen synovial sarcoma X breakpoint 2 (SSX-2) is classified as a cancer-testis antigen (CTA) based upon its tissue-restricted expression to germline cells and its frequent expression in a variety of malignancies. We have previously identified SSX-2 as a potential prostate cancer antigen that can be inducibly expressed in several prostate cancer cell lines. We have also previously identified two nonamer peptides from the amino acid sequence of SSX-2 that have significant affinity for HLA-A2 as assessed by in vitro T2 binding assay. These HLA-2-binding peptides were found to represent HLA-A2-restricted epitopes, able to induce peptide-specific immune responses in peptide- or DNA-immunized HHD-II (HLA-A2.1+/HLA-DR1+ transgenic) mice. Given these observations, the current study was conducted to determine if CTL generated in vaccinated HHD-II mice are capable of lysing HLA-A2+ prostate cancer cells and whether SSX-2 vaccine efficacy could be enhanced using an altered peptide ligand binding strategy. We found that peptides that were designed in silico to have increased or decreased HLA-A2+ affinity had enhanced and reduced HLA-A2 binding by in vitro T2 binding assay, respectively. Additionally, HHD-II mice immunized with modified SSX-2 vaccines developed increased or decreased frequencies of peptide-specific T cells in vivo, which could be detected by IFNγ ELISPOT assay and cytotoxicity assay. Animals immunized with enhancing modified SSX-2 vaccines generated CTL with cross-reactivity to the native SSX-2 epitopes. These CTL were capable of lysing target cells pulsed with both modified and native peptides and could also lyse an HLA-A2-expressing prostate cancer cell line. These results provide support for the further investigation of SSX-2 as an immunotherapeutic target for prostate cancer using vaccines modified to increase MHC class I binding.

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 2397.
Abstract 24: Labeling of oxidizable proteins with a photoactivatable analog of the antitumor agent DMXAA and evidence for redox signaling in its mode of action
Authors: Ching Lai-Ming , Wang Liang-Chuan S. , Brauer Romy , Bridewell David J.A , Henare Kimiora , Palmer Brian D.
Published: February 2011

DMXAA (5,6-dimethylxanthenone-4-acetic acid) is in late stages of clinical development as a tumor vascular disrupting agent. The signalling pathway(s) and molecular target(s) for this drug are still undefined. As an approach towards identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA is used in the studies here to probe for cellular binding proteins. Unexpectedly, over 20 proteins were photo-affinity labelled from the cytosolic protein extracts from murine spleen cells and the RAW 264.7 murine macrophage-like cell line. While no protein domain, fold or binding site for a specific ligand was found to be shared by all the candidate proteins, almost all were noted to be oxidisable proteins, implicating a role for redox cycling in the action of DMXAA. Consistent with this hypothesis, we showed that DMXAA caused an accumulative increase in concentrations of reactive oxygen species (ROS) in RAW 264.7 cells over the first two hours. This increase in ROS was suppressed in the presence of the anti-oxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. SiRNA knock-down in RAW 264.7 cells of one of the photolabelled proteins, superoxide dismutase 1, an ROS scavenger resulted in an increase in tumour necrosis factor-alpha production in response to DMXAA compared with controls treated with transfection reagent alone, or with unrelated siRNA molecules. Knock-down of two other photoaffinity labelled proteins which do not have direct ROS scavenging properties, thioredoxin and protein disulfide isomerase 6, had no significant effect on tumour necrosis factor-alpha production. The results from these three lines of study have led us to speculate on a role of modulation of redox signalling in cytokine induction by DMXAA.

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 24.
Abstract 2412: Sorafenib can be effectively combined with GM-CSF secreting cellular immunotherapy in a mouse model of HER-2+ breast cancer
Authors: Edwards Justin P. , Leatherman James M. , Goding Steven R. , Erdinc Melek M. , Emens Leisha A.
Published: February 2011

Due to the complexities of the tumor microenvironment and the frequent emergence of drug resistance with treatment, it is increasingly evident that cancer therapy will have to hit multiple host and tumor targets simultaneously. We previously reported that DC101, an antiangiogenic monoclonal antibody specific for the vascular endothelial growth factor receptor-2 (VEGFR-2), can enhance tumor immunity by a T cell-dependent mechanism. Sorafenib, a small molecule that also disrupts angiogenesis, blocks signaling through multiple kinases that control tumor growth and progression. It has also been reported to inhibit both dendritic cell (DC) and T cell function. To further explore the intrinsic tumor- and host-based effects of sorafenib, we tested it against murine HER-2+ breast tumor cells (NT) in vitro, and in NT-tumor-bearing FVB/N (immune-competent) and neu-N (immune tolerant to HER-2) mice in vivo. Sorafenib inhibited the growth of NT tumor cells in vitro, inducing apoptosis. Western blot analysis revealed that Sorafenib interfered with ERK/MAPK, but not AKT, signaling pathways, both of which are constitutively activated when HER-2 is over-expressed. Single agent sorafenib effectively controlled the growth of established tumors in immune competent FVB/N mice, but did not result in complete tumor clearance. In contrast, sorafenib alone did not significantly impact the outgrowth of established tumors in neu-N mice, where tumor-specific immune tolerance is in play. Consistent with this observation, depletion studies conducted in immune competent FVB/N mice revealed that the control of tumor growth is in part dependent on T cells, but not macrophages or NK cells. While sorafenib treatment alone did not induce immunity specific for RNEU420–429 (the immunodominant HER-2 epitope), it did accelerate tumor clearance induced by vaccination with a granulocyte-macrophage colony-simulating factor (GM-CSF)-secreting, HER-2+ cellular vaccine in tumor-bearing FVB/N mice compared to either drug treatment or vaccination alone. Finally, sorafenib neither augmented nor inhibited the vaccine-induced T cell response specific for RNEU420–429 as measured by ELISPOT and intracellular cytokine staining. Overall, these findings suggest that vaccine-based immunotherapy can interact with kinase inhibitors by distinct mechanisms for enhanced therapeutic response.

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 2412.