bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1223: Fibronectin induced lung cancer cell proliferation is dependent on induction of ILK through integrin B1-mediated activation of p38 MAPK and PI3-K
Authors: Sun XiaoJuan , Ritzenthaler Jeffrey D. , Zhong XiaoRong , Roman Jesse , Han Shouwei
Published: February 2011

Fibronectin is a multi-functional matrix glycoprotein protein that has been linked to tumor progression. We previously demonstrated that cellular fibronectin (cFN) increased non-small cell lung carcinoma (NSCLC) cell growth through signals mediated by the integrin α5β1. Here, we explore the role of integrin-linked kinase (ILK), an integrin β1 subunit cytoplasmic domain interactor in cFN induced cell growth. We found that cFN increases ILK expression and kinase activity, promotes protein-protein interactions between ILK and integrin β1. Silencing of ILK attenuated the effect of cFN on cell proliferation. Knockdown of the integrin β1 subunit, but not the integrin α5, blocked the effect of cFN on ILK expression. In addition, the inhibitor of p38MAPK, SB239063, and the inhibitor of PI3-K, wortmannin, abrogated the effect of cFN on ILK expression, whereas the inhibitor of ERK, PD98059, had no effect. Taken together, our results demonstrate that fibronectin induces the expression of ILK through integrin β1-mediated signals that include p38 MAPK and PI3-K. This in turn, promotes lung carcinoma cell proliferation.

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 1223.
Abstract 1228: HOXB13 promotes androgen independent growth of prostate cancer cells by the activation of E2F signaling
Authors: Kim Young-Rang , Oh Kyung-Jin , Choi Chan , Kim Min Soo , Kang Taek Won , Jung Chaeyong
Published: February 2011

Androgen signaling plays a critical role in the development of prostate cancer and its progression. However, androgen-independent prostate cancer cells emerge after hormone ablation therapy, resulting in significant clinical problems. We have previously demonstrated that the HOXB13 homeodomain protein functions as a prostate cancer cell growth suppressor by inhibiting androgen-mediated signals. However, the role of the HOXB13 in androgen-independent growth of prostate cancer cells remains unexplained. In this report, we first demonstrated that HOXB13 was highly overexpressed in hormone-refractory tumors compared to tumors without prostate-specific antigen after initial treatment. Functionally, in an androgen-free environment minimal induction of HOXB13 in LNCaP prostate cancer cells, to the level of the normal prostate, markedly promoted cell proliferation while suppression inhibited cell proliferation. The HOXB13-mediated cell growth promotion in the absence of androgen, appears to be mainly accomplished through the activation of RB-E2F signaling by inhibiting the expression of the p21waf tumor suppressor. Indeed, forced expression of HOXB13 dramatically decreased expression of p21waf; this inhibition largely affected HOXB13-mediated promotion of E2F signaling. Taken together, the results of this study demonstrated the presence of a novel pathway that helps understand androgen-independent survival of prostate cancer cells. These findings suggest that upregulation of HOXB13 is associated with an additive growth advantage of prostate cancer cells in the absence of or low androgen concentrations, by the regulation of p21-mediated E2F signaling.

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 1228.
Abstract 1231: Nuclear Factor I family members interact with FoxA1 to regulate androgen responsive promoters
Authors: Boachie Adu M. , Degraff David , Yu Xiuping , Sun Qian , Friedman David , Gronostajski Richard , Matusik Robert
Published: February 2011

Forkhead (Fox) proteins are an important class of transcription factors (TFs) that act as molecular “pioneers” by increasing chromatin accessibility. The ability of FoxA1 to act in this manner allows for the subsequent nucleation of responsive promoters by additional, tissue specific TFs, allowing for gene regulation. Our laboratory has previously shown that FoxA1 interacts directly with the androgen receptor (AR), and that the expression of FoxA1 is essential for normal prostate maturation and differentiation. Thus, the ability of FoxA1 to enter a transcriptional complex with AR and other TFs contributes to tissue-specific gene expression in the prostate, and determining the components of FoxA1/AR TF complexes will identify additional targets for the treatment of castrate-resistant PCa.

In order to identify additional proteins that interact with FoxA1, we ectopically expressed dual-affinity tagged FoxA1 in the human PCa cell line LNCaP. Following tandem purification and mass spectrometric analysis, we identified several FoxA1-interacting proteins, including Nuclear Factor I x (NFIx). NFIx is a member of the Nuclear Factor family of TFs, consisting of NFIa, NFIb, NFIc, and NFIx. Co-immunoprecipitation studies verify that NFI family members interact with FoxA1, and Chromatin immunoprecipitation (ChIP) experiments indicate NFI family members bind to androgen regulated, prostate-specific promoters. Finally, transient transfection experiments indicate that NFI family members modulate the androgenic stimulation of the Probasin and PSA promoters both in the presence and absence of FoxA1.

In summary, we have identified NFIx as a novel FoxA1 interacting protein. NFI family members act to modulate the transcriptional activity of FoxA1 and AR on prostate-specific target promoters.

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 1231.
Abstract 1234: Erk-dependent phosphorylation of Notch intracellular domain (NIC) correlates with increased NIC-dependent transcription
Authors: Tremblay Isabelle , Boucher Marie-Josee
Published: February 2011

Background. Recent studies have suggested that the ability of Ras to transform cells depends on cooperation with the Notch signaling pathway. However, the molecular and cellular mechanisms involved in this cooperation remain unknown. Aim. To evaluate the impact of the Mek/Erk pathway, downstream of Ras, on Notch signaling. Methods. HEK293T and human pancreatic cancer cells MIA PaCa-2 were used. Mek/Erk activity was down-regulated by treatment with the specific inhibitor U0126 (10 µM). Transfections or retroviral infections of a constitutive active form of KRas or Mek (MekCA) were used to increase Mek/Erk activity. NIC-dependent transcription was measured by luciferase assays using a specific (CSL) luciferase reporter gene. Results. 1- Transient transfection of an activated KRas stimulated by 1.7-fold NIC-dependent transcription. Transfection of MekCA increased by 4.3-fold NIC-dependent transcription while a wild-type form of Mek (MekWT) was without effect. In parallel, treatment with U0126 reduced by 60% the basal NIC-dependent transcription and completely blocked the KRas- and MekCA-induced NIC-dependent transcription. This negative effect of U0126 on NIC-dependent transcription correlated with decreased nuclear staining of the NIC target gene Hes1. 2- Western blot analysis revealed higher molecular weight forms of NIC when cells were transfected/infected with MekCA as compared to control MekWT-transfected/infected cells. U0126 treatment prevented the MekCA-induced decreased in mobility shift of NIC suggesting that NIC is subjected to Mek-dependent post-translational modifications in vivo. 3- Immunoprecipitation followed by in vitro phosphatase assay revealed that these MekCA-induced higher molecular weight forms of NIC were phosphorylated forms of NIC. 4- By in vitro kinase assays, we demonstrated that NIC worked as Erk substrate. 5- Finally, following in vivo phospholabeling, we observed that NIC immunoprecipitated from MekCA-transfected cells was more phosphorylated as compared to NIC immunoprecipitated from MekWT-transfected cells. Conclusion. Our results provide the novel discovery that Mek/Erk signaling promotes NIC phosphorylation which correlates with increased NIC-dependent transcription. Our discovery thus provides a possible cellular mechanism by which Ras and Notch cooperate to promote cellular transformation.

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 1234.
Abstract 1245: Polyribosome association of tyrosine phosphorylated RACK1 with activated Src regulates translation of specific proteins mediating breast cancer growth and metastasis
Authors: Scott Gary K. , Gabriel Bianca , Held Jason , Benz Christopher C.
Published: February 2011

Dysregulation of translational control, characterized by aberrant expression of proteins critical for growth and metastatic potential, is emerging as an essential aspect of tumorigenesis. Receptor for activated protein kinase C-1 (RACK1) is a scaffold protein known to coordinate the cellular localization of numerous signaling proteins including PKC and Src, and has recently been identified as an essential ribosome component binding to the head region of the 40S subunit. RACK1 was identified in a mass spectrometry screen of polyribosome proteins from human breast cancer cells demonstrating altered mobility on 2D gel electrophoresis following growth-arresting doses of a histone deacetylase (HDAC) inhibitor which suggested that post-translational modifications of ribosome associated RACK1 might selectively influence protein synthesis and cancer cell growth. Polyribosome profiling of MDA-231 and SKBR3 breast cancer cells revealed that while the entire complement of lysate soluble RACK1 was ribosome associated in both cell lines, only RACK1 from the MDA-231 cells appeared significantly tyrosine phosphorylated. Furthermore, tyrosine phosphorylated Src (pTyr-416), while 10 fold more abundant in the SKBR3 untranslated fractions relative to those from MDA-231, was associated with the polyribosome factions of MDA-231 but not those of SKBR3, consistent with previous observations linking activated Src with tyrosine phosphorylated RACK1. Lentiviral delivery of shRNA was used to investigate the functional consequences of selective RACK1 knockdown. In MDA-231 cells, efficient RACK1 knockdown produced complete growth arrest and loss of motility without induction of apoptosis or cell senescence; however, this growth arrested phenotype is associated with near complete loss of cyclin D1 and TGFbetaR1 expression without change in the expression of housekeeping proteins beta-actin and GAPDH. Taken together, these results implicate tyrosine phosphorylated RACK1 in association with polysomal pTyr-Src as translational activators of specific proteins mediating breast cancer proliferation 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 1245.
Abstract 1248: 14-3-3 interacts with TC1(C8orf4) to protect the protein from rapid degradation and regulates cancer cell invasiveness
Authors: Lee Inchul
Published: February 2011

14-3-3 interacts with TC1(C8orf4) to protect the protein from rapid degradation and regulates cancer cell invasiveness.

Jungtae Kim, Yusun Jung, Dong Wook Kim, Hyunju Lee, Kyuyoung Song, Inchul Lee. Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Republic of Korea

TC1(C8orf4) is a multifunctional regulator associated with aggressive biological behavior of cancer and poor survival of patients (Clin Cancer Res 2006;12:3541-8). It regulates the Wnt/β-catenin pathway (Cancer Res 2006;66:723-8).and induces endothelial inflammation (J Immunol 2009;183:3996-4002). TC1 is up-regulated by NF-kB (FEBS letters 2006;580:3519-24), and heat shock and other cellular stress (Biochem Biophys Res Commun 2007;360:447-52).

TC1 is a small protein present in vertebrates, which is degraded rapidly. TC1 interacted with 14-3-3β upon yeast two hybrid screening. The interaction of TC1 with 14-3-3β and 14-3-3ε was confirmed in HEK293T cells upon cotransfection. The deletion mutant analysis showed that the N-terminus of TC1 and C-terminus of 14-3-3 interacted with each other. The binding site of TC1 appeared to involve the Ser15 residue. The interaction was inhibited by serum starvation, staurosporine, and GSK3β inhibitor, indicating the requirement of TC1 phosphorylation, probably of Ser15, for the interaction. The half-life of transfected HA-TC1 was 2.49 hours in HEK293T cells, while it was elongated to 5.07 hours upon co-transfection with 14-3-3β. 14-3-3 knock down reduced TC1 expression. Our data together suggested that 14-3-3 protected TC1 in vivo.

14-3-3 proteins have been implicated in the regulation of various biological functions. However, the role in cancer is not clear. TC1 was shown to enhance the Matrigel invasiveness of cancer cells (Clin Cancer Res 2006;12:3541-8). 14-3-3 knock down using si14-3-3β reduced the Matrigel invasiveness of HeLa cells significantly (p<0.0001). Cancer cell proliferation was not affected by 14-3-3 transfection or knock down. Our data suggest the requirement of 14-3-3 in cancer cell invasiveness potentially by regulating TC1.

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 1248.
Abstract 1250: Inhibition of FAK and engagement of migration leads to similar changes in patterns of transcription factor activation in glioma cells
Authors: Berens Michael E. , Tran Nhan L. , Loftus Joseph C.
Published: February 2011

Malignant dispersion of glioma cells is a nemesis to clinical management, provoking the need for a better understanding of the underlying molecular drivers of invasion. The changes in signal transduction and consequent alterations in activation/de-activation of Transcription Factors (TFs) in glioma cells were investigated coincident to migration-engagement or impaired FAK kinase function by small molecule inhibition (PF-562,271). Earlier work showed that FAK and Pyk2 affect proliferation and migration in reciprocal ways in glioma cells (Lipinski CA, et al 2008; Loftus JC, et al 2009). The interaction of FAK and Pyk2 with specific downstream signaling effectors and the resultant activation of disparate signaling pathways is likely to script a signaling “signature” that will reveal to what extent glioma cell malignant behaviors are dependent on these kinases. Inhibition of FAK leads to diminution of signaling by pSrc, pJnk, and pNFkB, and variable activation of pAkt, and pp38. Of the 20 TFs assayed (Marligen, Inc.), the activation state of 7 TFs showed significant and consistent patterns of change across 6 primary GBM cell strains following FAK inhibition. Across a panel of 6 human GBM xenograft lines SP1, p53, E2F1-5, and NFκB evidenced strong activation (heightened nuclear localization), while AR (androgen receptor), Myc-Max, and CREB activation levels dropped by more than 2-fold following PF-562,271 treatment. Separate studies of established glioma cell lines under conditions of sparse plating (migration) and of dense crowded plating (migration-constrained), showed that p53, E2F1-5, and NFκB were standouts for activation accompanying migration, also associated by strong reduced activation of AR, Myc-Max, and CREB. (Because glioma cells following FAK knockdown or FAK inhibition show reduced entry into cell cycle, the signaling linkage between FAK and Myc-Max activation and also between FAK signaling and CREB activation suggest mechanistic ties to phenotype. E2F1 is a transcription factor that regulates genes involved in cell proliferation and cell survival and has even been suggested to fit a paradigm of oncogene addiction in glioma cells. Our findings demonstrate phenotypic responses in glioma cells downstream of FAK signaling linked to the activation or inactivation of specific transcription factors. If these patterns of TF activation state, and the signaling pathways upstream of that activation, associate (or account for) patterns of glioma cell invasion or proliferation in situ, they will suggest points of vulnerability in the underlying pathological processes of this disease. Supported by NIH NS042262 (Berens) and NIH CA103956 (Loftus).

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 1250.
Abstract 1251: Increased expression of the plasminogen activator system due to differential protease-activated receptor signaling in breast cancer cells
Authors: McEachron Troy , Church Frank , Mackman Nigel
Published: February 2011

Activation of protease activated receptor-1 and −2 (PAR-1 and PAR-2) on tumor cells by coagulation proteases, such as factor Xa (FXa) and thrombin, leads to the expression of urokinase plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1). uPA has been shown to contribute to malignancy by increasing tumor cell migration, survival, extracellular matrix degradation, and angiogenesis. The goal of this study was to determine how activation of PAR-1 and PAR-2 by coagulation proteases regulates uPA and PAI-1 expression. We used the 4T1 mouse mammary adenocarcinoma cell line as a model because it expresses both PAR-1 and PAR-2. Using shRNA technology, we made derivatives of the 4T1 cells with reduced expression of either PAR-1 or PAR-2. 4T1 cells carrying a non-targeting shRNA construct were used as negative controls. We found that FXa and thrombin induced uPA expression in the control and the PAR-2 shRNA cells, but not in the PAR-1 shRNA cells. In contrast, FXa treatment did not increase PAI-1 levels in the control, PAR-1 shRNA, or PAR-2 shRNA cells. Thrombin was able to induce PAI-1 in the control cells, but not in the PAR-1 shRNA or the PAR-2 shRNA cells. Taken together, our data suggests that uPA expression is dependent on PAR-1 activation whereas PAI-1 expression requires a PAR-1/PAR-2 heterodimer.

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 1251.
Abstract 1254: Kaposi's Sarcoma herpesvirus K1's transcellular inhibition of Fas-mediated apoptosis
Authors: Daniluk Urszula , Berkova Zuzana , Wise Jillian Frances , Tao Rong-Hua , Hossein Rezaeian Abdol , Wang Shu , Prakash Om , Samaniego Felipe
Published: February 2011

The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas is not known. K1 is known to activate Akt and nuclear factor kappa B (NF-kB) through immunoreceptor tyrosine-based activation motif (ITAM) and may also bind to Fas receptor through its immunoglobulin (Ig) chain-like domain and interfere with apoptosis.

We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by altered Fas signaling.

Examination of mice expressing K1 via a ubiquitous promoter showed that 90% K1 transgenic mice (n=10) had developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. In vitro overexpression of an Ig domain-containing protein CD79b or treatment cells with K1 peptides revealed competition with K1-Fas binding in a dose-dependent manner and rate enhancement of Fas-mediated apoptosis. We have also shown that K1 suppressed Fas-mediated apoptosis, even in cells that did not express K1. Transfection of K1 into one pool of mouse cells protected against Fas-mediated apoptosis of a second pool of human Fas-transfected mouse cells indicating protection in trans.

This analysis indicates a key role of K1 in suppression of Fas-mediated apoptosis which operates in a cis and trans protective role against apoptosis.

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 1254.
Abstract 1260: Analysis of death receptor 5 and caspase-8 expression in primary and metastatic head and neck squamous cell carcinoma and their prognostic impact
Authors: Elrod Heath A. , Fan Songqing , Muller Susan , Chen Georgia Z. , Pan Lin , Tighiouart Mourad , Shin Dong M. , Khuri Fadlo R. , Sun Shi-Yong
Published: February 2011

Death receptor 5 (DR5) and caspase-8 are major components in the extrinsic apoptotic pathway. The alterations of the expression of these proteins during the metastasis of head and neck squamous cell carcinoma (HNSCC) and their prognostic impact have not been reported. The present study analyzes the expression of DR5 and caspase-8 by immunohistochemistry (IHC) in primary and metastatic HNSCCs and their impact on patient survival. Tumor samples in this study included approximately 90 primary HNSCC with no evidence of metastasis and approximately 90 primary HNSCC and their matching lymph node metastasis. IHC analysis revealed a significant loss or downregulation of DR5 expression in primary tumors with metastasis [weight index (WI) = 221 ± 64.9 (mean ± SD), n = 92] and their matching lymph node metastasis (WI = 224 ± 71.6, n = 85) compared to primary tumors with no evidence of metastasis (WI = 250 ± 43.9, n = 94) (P < 0.01). A similar trend was also observed in caspase-8 expression although it is not statistically significant. Downregulation of caspase-8 and DR5 expression was significantly correlated with poorly differentiated tumors compared to moderately and well differentiated tumors (P < 0.05). Univariate analysis indicates that, in HNSCC with no metastasis, higher expression of caspase-8 significantly correlated with better disease-free survival and overall survival (P < 0.05 or 0.001) if mean value is used as a cutoff. However, in HNSCC with lymph node metastasis, higher caspase-8 expression significantly correlated with poorer disease-free survival and overall survival (< 0.05 or 0.01). Similar results were also generated when we combined both DR5 and caspase-8. Moreover, multivariable analysis indicates that in patients without lymph node metastasis, high caspase-8 is significantly associated with better survival after adjusting age and stage (P < 0.0001). Together, we suggest that both proteins may be involved in regulation of HNSCC metastasis. (G.C., D.M.S., F.R.K. and S-Y.S. are Georgia Cancer Coalition Distinguished Cancer Scholars. This study was supported by funds from the Georgia Cancer Coalition Distinguished Cancer Scholar and NIH Head and Neck SPORE P50 CA128613 awards)

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 1260.
Abstract 1266: TNF-alpha mediated down-regulation of Na+/H+ exchanger 1 (NHE1) in breast cancer MCF7 cells and its relevance to apoptosis
Authors: Chua Sui Huay , Clement Marie V.
Published: February 2011

Intracellular alkalinization is a hallmark of cancer cells shown to confer resistance against caspase mediated apoptosis. Na+/H+ exchanger 1 (NHE1), an ion exchanger important in the maintenance of intracellular pH and cell volume homeostasis had been implicated in this intracellular alkalinization. Increased NHE1 protein level and activity has been found in many cancer cells types where increase in intracellular pH by constitutively active NHE1 confers resistance to apoptosis in these cells. Silencing of NHE1 protein in cancer cells results in reduced cell growth and increased in sensitivity to death triggers. TNF-α can induce H2O2 production in cells to mediate apoptosis with characteristic cell shrinkage. H2O2 had recently been shown to downregulate NHE1 expression and increase cells’ sensitivity to apoptosis. This study was undertaken to study the role of caspases and H2O2 in the regulation of NHE1 protein and gene expression by TNF-α. Results showed that TNF-α had both cytostatic and cytotoxic effects on MCF-7 cells with concurrent decrease in intracellular pH and NHE1 activity. NHE1 was found to be downregulated both at the protein and mRNA level following TNF-α treatment. Gelshift assay showed that transcription factors binding to the promoter of NHE1 could be blocked by pre-incubation with H2O2, suggesting that transcription of NHE1 could be redox-regulated. Indeed, we also show by using a luciferase reporter assay that NHE1 promoter is down-regulated in presence of H2O2. Various caspases were activated in TNF-α treated cells and the inhibition of caspases prevented the downregulation of NHE1. ROS was elevated in MCF-7 within 2 hours of treatment. Incubation of cells with catalase increased their survival in response to TNF-α, thus implicating H2O2 as a mediator of TNF-α induced cell death. Overexpression of catalase in MCF-7 reduced the downregulation of NHE1 by TNF-α. Our results suggest by TNF-α mediate cell death through decreasing NHE1 activity and down-regulating NHE1 expression in a ROS mediated manner.

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 1266.
Abstract 1268: Identification of a novel tumor necrosis factor receptor-associated factor 6-binding partner that is a potential lysine-63 linked ubiquitination substrate
Authors: Huang Chien-Ling , Cheng Ju-Chien , Liu Shau-Yu , Tseng Ching-Ping
Published: February 2011

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin E3 ligase that catalyzes lysine-63 linked polyubiquitination of various signaling proteins. The recruitment and formation of different protein complexes with TRAF6 leads to activation of TRAF6-associated signaling and regulates cell proliferation and apoptosis. In this study, we search for novel TRAF6-binding partners that may have implication in TRAF6 signaling and cancer progression. Bioinformatic analysis identified a putative TRAF6-binding protein (TRAF6BP) that contains the consensus TRAF6-binding motif X-X-P-X-E-X-X-Ar/Ac-X and thereby TRAF6BP was subjected to further characterization. In accord with the bioinformatics prediction, coimmunoprecipitation analysis demonstrated the binding between TRAF6BP and TRAF6. TRAF6BP also interacts with other TRAF proteins including TRAF-2, −3, −5 but the interaction was much weaker than TRAF6. Domain mapping with TRAF6 deletion mutants identified the TRAF-C domain of TRAF6 is important for its interaction with TRAF6BP. In vivo ubiquitination assay further unveiled that TRAF6BP is a substrate of TRAF6 E3 ligase and underwent ubiquitination in the presence of TRAF6. Only the expression of full-length TRAF6, but not the TRAF-C deletion mutants of TRAF6 resulted in the ubiquitination of TRAF6BP. In addition, TRAF6BP ubiquitiation was abrogated when the lysine-63 ubiquitin mutant (K63R) but not the lysine-48 mutant (K48R) was used as the ubiquitin substrate. Together, we demonstrated for the first time that TRAF6BP is a novel TRAF6-binding protein and is a potential lysine-63 ubiquitination substrate of TRAF6; the TRAF6BP-TRAF6 interaction is essential for TRAF6 to ubiquitinate TRAF6BP. The significances of TRAF6BP-TRAF6 interactions and TRAF6BP ubiquitination in cancer progression and TRAF6 signaling are worthy to further investigation.

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 1268.
Abstract 1269: Molecular changes underlying bortezomib sensitization of cancer cells to TRAIL-mediated apoptosis
Authors: Brooks Alan D. , Thompson Candace R. , Sayers Thomas J.
Published: February 2011

We have previously shown that the proteasome inhibitor bortezomib (Velcade/PS-341) specifically sensitized certain human renal carcinoma (RCC) lines to apoptosis induced by the TNF family-member TRAIL. A comparison of RCC sensitized by bortezomib with those that remained resistant revealed no major bortezomib-induced changes in the levels of many apoptosis proteins. Also, when bortezomib treatment induced specific changes in protein levels (mcl-1, Bik and survivin) these changes did not directly correlate with subsequent sensitization or resistance to TRAIL apoptosis. In addition changes in surface expression of TRAIL receptors (DR4, DR5, DcR1, DcR2) following bortezomib treatment also did not correlate well with sensitization or resistance. By contrast, enhanced procaspase 8 activation following bortezomib pretreatment and TRAIL exposure was only observed in the sensitized RCC. This increase in procaspase 8 activation in cell extracts of sensitized RCC was observed using both enzyme assays as well as western blotting for activated (cleaved) caspase 8. Use of the apoptosis-resistant MCF-7 cell line demonstrated that bortezomib pretreatment increased caspase-8 activation on exposure to TRAIL even in the absence of subsequent downstream caspase activation and apoptosis. This suggests that bortezomib increases caspase-8 activation upstream at the death-inducing signaling complex (DISC). Furthermore, immunoprecipitation of the DISC demonstrated higher levels of active caspase 8 in the DISC of sensitized but not resistant RCC. Interestingly ACHN (a bortezomib-sensitized RCC) was also sensitized to TRAIL apoptosis when adherence was prevented by coating cell culture dishes with poly-HEMA. In parallel this loss of adherence abolished the sensitizing effects of bortezomib treatment. Bortezomib and poly-HEMA treatment of RCC both result in reduced cellular proliferation, and may change cell adherence properties. Whether both of these treatments sensitize RCC to TRAIL using common or distinct molecular pathways is under further investigation.

Funded in part by DHSS #NO1-CO-12400

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 1269.
Abstract 1272: Targeting glioma stem cells through death receptor activation by AdsTRAIL
Authors: Puduvalli Vinay K. , Liu Yuanfang , Colman Howard , Lang Frederick , Xu Jihong
Published: February 2011

Emerging data suggests that human malignant gliomas may be sustained by the activity of a subpopulation of cells which have stem like characteristics; these glioma stem cells (GSC) exhibit resistance to conventional treatments and constitute novel targets for therapeutic strategies against these malignancies. The goal of this study was to examine whether GSC could be targeted using AdsTRAIL, an adenoviral vector expressing soluble TRAIL and to determine if AdsTRAIL effects could be potentiated by suppressing inhibitors of TRAIL-induced apoptosis. Analysis of expression of TRAIL activated death receptor pathway components showed that DR4, DR5, FADD and Caspase 8 were expressed in the majority of GSC although some cell lines expressed only one of the death receptors (DR4 or DR5). Several cell lines also showed high level of expression of survivin, an anti-apoptosis protein. Treatment of GSC, cultured as tumor spheres in stem cell medium, with AdsTRAIL resulted in dissociation of spheres into a single cell suspension and induction of apoptosis dose and time dependent manner, changes which were not seen in the AdEGFP control; no morphologic changes or induction of apoptosis was seen in GSC spheres treated with purified soluble TRAIL. To determine if induction of apoptosis by AdsTRAIL could be enhanced by suppressing inhibitors of TRAIL induced apoptosis, GSC were treated with AdXAF1, an adenoviral construct expressing XAF1, an endogenous inhibitor of XIAP, prior to exposure to AdsTRAIL. Viability was monitored by expression of the fluorescent marker EGFP in the treated cells. The combination of AdsTRAIL and AdXAF1 potently induced apoptosis to a greater degree than either agent alone. These results suggest that despite their resistance to conventional therapeutic agents such as chemotherapy and radiation, glioma stem cells could potentially be targeted by apoptosis inducing biological agents such as AdsTRAIL. Further characterization of the effects of AdsTRAIL against GSC is required to fully assess the potential of this agent against treatment resistant malignant gliomas.

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 1272.
Abstract 1278: Simultaneous downregulation of uPAR and cathepsin B inhibits angiogenesis and migration and induces caspase 8-mediated apoptosis in U87 stem cells and 4910 glioma xenograft stem cells
Authors: Gopinath Sreelatha , Malla Rama Rao , Gondi Christopher S. , Fassett Daniel , Klopfenstein Jeffrey D. , Dinh Dzung H. , Gujrati Meena , Rao Jasti S.
Published: February 2011

Malignant gliomas are characterized by invasive and infiltrative behavior. Gliomas are considered to be heterogeneous bulk tumors that comprise differentiated and undifferentiated cells with self-renewal and partial differentiation capabilities. These glioma-initiating cells (GIC) are responsible for the initiation and recurrence of gliomas even after various treatments. As such, novel therapies should target GIC. Cathepsin B and urokinase-type plasminogen activator receptor (uPAR) are overexpressed in gliomas, including GIC. We isolated GIC from U87 (U87S) and 4910 (4910S) by sorting the cells for both CD133 and STRO-1 by FACS analysis. The stem cell nature of these cells was confirmed by neurosphere formation assay and western blot analysis for CD133 and STRO-1 expression. GIC cells expressed 2 to 3-fold increased levels of CD133 and STRO-1 as compared to glioma cells (U87N and 4910N). By isolating and utilizing GIC, we demonstrate that downregulation of uPAR (pU) and cathepsin B (pC) individually and simultaneously (pCU) lead to remarkable inhibition of angiogenesis and migration and activated caspase 8-mediated apoptosis in GIC and U87N and 4910N cells. The treatment retarded the expression of pro-angiogenic molecules in both stem cells and glioma cells but responded differently in terms of expression of certain molecules, such as angiogenin and TIMP-1. Similarly, an apoptotic antibody array revealed that stem cells and glioma cells respond differently to pCU treatment, but both sets of cells exhibited increased expression of pro-apoptotic molecules. This induction of apoptosis was also confirmed by cleavage of caspase 8, caspase 3 and PARP as well as TUNEL assay. Similarly, spheroid migration and matrigel invasion assays, revealed that downregulation of uPAR and cathepsin B inhibited cell migration by 70-75% and 45-50% and invasion by 75-80% and 45-50% in GIC and normal glioma cells, respectively. Untreated glioma stem cells were more migratory (∼30%) and invasive (30-40%) as compared to normal glioma cells. In vivo studies showed few to no tumor cells in brain sections of mice implanted with either GIC or normal glioma cells treated with pCU as determined by H&E staining. These results indicate the potential of RNAi-mediated downregulation of uPAR and cathepsin B in developing new therapeutics for GIC in particular and gliomas in general.

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 1278.
Abstract 1285: The effects of the HK5-ferritin interaction and iron on tumor angiogenesis
Authors: Huhn Annissa J. , Parsonage Derek , Torti Suzy V. , Hollis Thomas
Published: February 2011

Angiogenesis, or the development of new blood vessels from existing blood vessels, is a process essential to wound healing, reproduction, and tumor growth. Tumor angiogenesis is an important factor in the growth and spread of cancer. In order for tumors to grow beyond 2mm^3, vasculature is necessary to provide oxygen and nutrients and remove waste products. The imperfect blood vessels built within tumors also allow tumor cells to travel through the bloodstream and metastasize. Endogenous inhibitors of angiogenesis are known to form from plasma proteins. For example, angiostatin is derived from plasmin. A newly described angiogenesis inhibitor derived from high molecular weight kininogen (HK) is domain 5 of HK (HK5). HK5 has been shown to inhibit endothelial cell proliferation, migration and tube formation. HK5 binds to the endothelial cell surface receptors uPAR, cytokeratin-1, gC1qR and tropomyosin to disrupt angiogenic signaling. Our laboratory has shown that the iron storage protein ferritin binds to HK5, inhibiting its antiangiogenic effects and thereby allowing endothelial cells to migrate and proliferate. We have begun to investigate the physical interaction of ferritin with the HK5 protein with the long-term goal of developing inhibitors of the HK5-ferritin interaction. Human recombinant HK5 and ferritin H were purified and their interaction studied using fluorescence anisotropy. The two proteins were found to bind with a Kd of 1.36μM. The influence of metals on the interaction was also evaluated. We found that in the presence of iron the interaction between ferritin and HK5 was maximal, while in the presence of zinc, the interaction was minimal. EDTA pretreatment of the two proteins further improved binding. The results suggest that iron is essential to the optimal binding interaction of HK5 and ferritin. Future studies will be directed at the identification of specific amino acid residues involved in this interaction. (supported by NIH R01 DK071892)

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 1285.
Abstract 1289: The recombinant kringle domain of urokinase plasminogen activator (uPA) inhibits VEGF165-induced proliferation of HUVEC cells
Authors: Kim Byeong Mo , Joe Young Ae , Hong Sung Hee
Published: February 2011

Angiogenesis is associated with tumor growth, invasion and metastasis. Many growth factors and cytokines are engaged in angiogenesis. Among them, vascular endothelial growth factor (VEGF) is one of the most potent and specific angiogenic factors and stimulates endothelial cell proliferation, protease expression, migration and subsequent differentiation to form new vessels. It has been previously revealed that the recombinant kringle domain of urokinase plasminogen activators (uPA) (UK1) exerts antiangiogenic activity against VEGF-induced angiogenesis using in vitro or in vivo model. In this study, we explored molecular signaling mechanisms involved in this activity. The antiangiogenic mechanism of UK1 was investigated in vitro by measuring phosphorylation levels of VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF/VEGFR2 signal molecules, and pre-treatment with UK1 blocked not only VEGF-induced phosphorylation of VEGFR2 but also phosphorylations of downstream signaling proteins, such as Erk and Akt. In addition, VEGF induced secretion of MMP2 in HUVEC, which efficiently suppressed by UK1 pre-treatment. However, mRNA levels of MMP2 as well as of MMP9, Timp1 and Timp2 remained unaffected by UK1. Overall, our findings suggest that UK1 inhibits VEGF-induced proliferation and migration of HUVEC by suppressing VEGF/VEGFR2-mediated angiogenic signals and MMP2 activity.

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 1289.
Abstract 1295: Inhibition of platelet derived growth factor receptor tyrosine kinase reduces lymphangiogenesis of human gastrointestinal cancer
Authors: Kodama Michiyo , Kitadai Yasuhiko , Sumida Tomonori , Ohnishi Mayu , Ohara Eiji , Shinagawa Kei , Tanaka Miwako , Tanaka Shinji , Yasui Wataru , Chayama Kazuaki
Published: February 2011

Objective: Vascular endothelial growth factor (VEGF)-C/D induces lymphangiogenesis by activating VEGF receptor (VEGFR)-3, which is expressed on lymphatic endothelial cells. Platelet derived growth factor receptor (PDGF-R) has also been detected on lymphatic endothelial cells and PDGF-BB acts as a lymphangiogenic factor in murine fibrosarcoma. We have previously reported that PDGF-R is expressed by lymphatic endothelial cells in human gastric cancer and its expression levels in tumor tissue correlate with lymph node metastasis. In this study, we examined whether inhibition of PDGF-R tyrosine kinase signaling by imatinib affects lymphangiogenesis and lymph node metastasis of human gastrointestinal cancer cells growing in the orthotopic site of nude mice. Methods: TMK-1 human gastric cancer cells or KM12SM human colon cancer cells were injected into the orthotopic site of nude mice. Groups of mice (n=10) received saline (control), imatinib, the cancer chemotherapeutic irinotecan, or a combination of imatinib and irinotecan. The lymphatic vessels were then stained with antibodies against Lyve 1. Results: Lymphatic endothelial cells as well as activated fibroblasts in the tumor stroma express PDGF-R. Four weeks of treatment with imatinib and irinotecan significantly inhibited lymph node metastasis (relative to control or single-agent therapy). Imatinib alone or in combination with irinotecan significantly decreased number and area of lymphatic vessels in the tumor stroma. Conclusion: Administration of a PDGF-R tyrosine kinase inhibitor in combination with irinotecan inhibits the lymph node metastasis of orthotopically implanted gastrointestinal cancer cells in nude mice by blocking PDGF-R signaling in tumor-associated lymphatic endothelial 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 1295.
Abstract 1298: Mechanisms of endothelial trophic support of genitourinary tumors
Authors: Liang June , Tilki Derya , Kobayashi Hideki , Rafii Shahin , Seandel Marco
Published: February 2011

Background: Classically, principal roles of the vascular endothelium have been considered to be support of oxygenation, nutrient delivery, waste product removal, and cellular trafficking. But the endothelium may also release paracrine signals to neighboring cells. Targeting such interactions could lower the threshold for sensitivity to conventional therapies, if pro-survival signals are blocked. We hypothesize that a subset of tumor cells are protected by endothelial-derived NF-kappa B (NF-KB)-mediated trophic signals that enhance tumor growth. Due to the confounding effects of circulating signals, it has been difficult to address these hypotheses using animal models. In contrast, in vitro models are limited by the requirement of primary endothelial cells (PECs) for serum and/or growth factors (GFs), complicating efforts to identify novel secreted factors. We employed a strategy to maintain PECs without serum by expressing the non-transforming adenoviral E4ORF1 gene. Under these conditions, E4ORF1-expressing (E4) PECs retain features of native tumor endothelium, including responsiveness to endothelial GFs and the ability to form functional vessels in vivo. Using solid tumor cell lines, including testicular germ cell tumor (GCT) and prostate cancer (PC) lines, we tested the ability of E4 PECs to support solid tumor progression. Methods: Constitutive expression of E4ORF1 in primary human umbilical vein endothelial cells (HUVECs) was achieved using lentivirus. Co-culture assays in serum-free media were used to test the effect of E4 PECs on cancer cell survival, proliferation and clonagenicity. Xenografts were performed by delivering cancer cells subcutaneously in the presence or absence of E4 PECs. Results: We first identified minimal media in which significant cancer cell proliferation was not observed in the absence of feeders. Under these conditions, we observed massive expansion of cell numbers for certain lines (e.g., 27×1) in the presence of E4 PECs (62.6+/-3.1 fold [mean+/-s.d.] for 27×1) vs. feeder-free control (2.2+/-0.1 fold) over 4 days. Changes in expression of stem cell-related genes in tumor cells during exposure to E4 PECs were examined to determine the frequency of stem-like cells in the culture. Results of co-inoculation studies using tumor cells in NOD-SCID mice will be discussed. Interventions to address the role of endothelial inside-out NF-KB signaling are on-going. Conclusions: Under stressful microenvironmental conditions, some but not all solid tumor cell lines require endothelial-derived growth and survival signals. Targeting these signals may lead to alternate strategies for eliminating residual tumor present after optimal therapy.

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 1298.