bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4475: IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor
Authors: Murugan Poongkavithai Vadevoo Sri , Rangaswamy Gunassekaran Gowri , Chi Lianhua , Padmanaban Guruprasath , Baek Moon-Chang , Kim In-San , Park Rang-Woon , Lee Byung-Heon
Published: May 2015

Targeted delivery of imaging agents and therapeutics to tumors would provide early detection and increased therapeutic efficacy against cancer. Using phage displayed-random peptide libraries, we have identified IL4RPep-1 (IL-4 receptor-binding peptide-1), CRKRLDRNC, that binds to IL-4 receptor (IL4R). IL4R is over-expressed on many types of cancer cells including lung cancer and breast cancer. Peptides have smaller size and in turn may exert better tissue penetration than bulky antibodies. IL4RPep-1 bound to H226 lung tumor cells that over-express IL4R, while little binding was observed in H460 lung tumor cells that express IL4R at low levels. When injected intravenously into nude mice bearing a subcutaneous H226 tumor, Cy7.5 near-infrared fluorescence (NIRF) dye-conjugated IL4RPep-1 selectively homed to tumor. IL4RPep-1-labeled liposomes containing doxorubicin showed more efficient anti-tumor growth activity than untargeted liposomes. In addition, IL4RPep-1-labeled, branched-type polyethyleneimine nanoparticles containing Bcl-xL siRNA more efficiently knocked down Bcl-xL expression and showed higher cytotoxic effect on cancer cells over-expressing IL4R than cancer cells expressing IL4R at low levels. These results suggest that IL4RPep-1 could be a useful probe for affinity-based imaging and drug delivery to tumor that over-expressing IL4R.

Citation Format: Poongkavithai Vadevoo Sri Murugan, Gunassekaran Gowri Rangaswamy, Lianhua Chi, Guruprasath Padmanaban, Moon-Chang Baek, In-San Kim, Rang-Woon Park, Byung-Heon Lee. IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4475. doi:10.1158/1538-7445.AM2014-4475
Abstract 4520: Inhibition of melanoma growth by small molecules that attenuate PI3K/PDK1 signaling and promote ATF2 mitochondrial localization
Authors: Varsano Tal , Feng Yongmei , Claps Giuseppina , Scortegagna Marzia , Lau Eric , Leonard Marilyn , Pinkerton Anthony , Pellecchia Maurizio , Davies Michael , Bosenberg Marcus , Ronai Ze'ev
Published: May 2015

Phosphoinositide-dependent kinase-1 (PDK-1) phosphorylates members of the AGC kinase superfamily, including AKT and PKC. We recently demonstrated the importance of PDK1 in melanoma development and progression (Oncogene 2013). Genetic ablation of PDK1 in melanocytes effectively delayed melanoma development and inhibited metastasis in a Braf/Cdkn2a model with WT or mutant Pten. In an effort to identify small molecule inhibitors that could affect the PDK1 signaling we characterized SBI-0089410, a small molecule identified in a screen for nuclear exclusion of the transcription factor ATF2, which attenuates its oncogenic activity while enabling its function at the mitochondrial membrane to promote apoptosis. SBI-0089410 reduced melanoma cell viability, inhibited colony formation and spheroid growth, decreased mitochondrial membrane permeability, decreased ATF2 phosphorylation by PKCϵ and altered ATF2 transcriptional activity. Notably, the effect of SBI-0089401 was attenuated by the overexpression of constitutively active PKCϵ or ATF2T52E, which carries a phosphomimetic mutation at the PKCϵ phosphorylation site. Reverse phase protein array (RPPA), validated by western blot analysis, revealed that SBI-408910 attenuates PI3K/AKT/PDK1 signaling pathways. In vivo, SBI-089410 attenuated the growth of Braf/Cdkn2a/Pten tumors in a syngeneic B6 mouse model, as well as prevented their resistance to BRAFi. Our studies substantiate the importance of targeting PI3K/PDK1 signaling in melanoma as a means for effective therapy and prevention of chemoresistance.

Citation Format: Tal Varsano, Yongmei Feng, Giuseppina Claps, Marzia Scortegagna, Eric Lau, Marilyn Leonard, Anthony Pinkerton, Maurizio Pellecchia, Michael Davies, Marcus Bosenberg, Ze'ev Ronai. Inhibition of melanoma growth by small molecules that attenuate PI3K/PDK1 signaling and promote ATF2 mitochondrial localization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4520. doi:10.1158/1538-7445.AM2014-4520
Abstract 458: Regulation of HIF1a by Set9 lysine methyltransferase
Authors: Liu Qiong , Geng Hao , Xue Changhui , Beer Tomasz M. , Qian David Z.
Published: May 2015

HIF-1 is consisted of an oxygen-sensitive HIF-1α and a constitutive HIF-1β subunit. The activity of HIF-1 is implicated in human cancer development. In normoxia, the HIF-1α subunit is constantly synthesized, but rapidly degraded by the oxygen-dependent mechanism. In hypoxia due to tumor growth and anti-angiogenic therapy, HIF-1α is stabilized and dimerizes with HIF-1β to form HIF-1, which transactivates genes promoting cancer hallmarks, including angiogenesis, metabolic reprogramming, migration and invasion. Notably, HIF-1 increases multiple genes on the glycolytic pathway, which mediates the metabolic adaptation of hypoxic stress. Therefore, HIF-1α is an intriguing anticancer target. However, how HIF-1α is regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase Set9 promotes HIF-1α protein stability in hypoxia and plays an important role in cancer cell glycolytic adaptation. Specifically, Set9 interacts with HIF-1α to maintain its stability in hypoxia. Silencing of Set9 by siRNA in hypoxia significantly reduces HIF-1α protein level, reduces HIF-1 transcriptional activity, and attenuates expressions of HIF-1-responsive glycolytic genes as well as hypoxia-induced glycolysis. Further, we have found that Set9 binds to the chromatins of hypoxia response elements within HIF-1-responsive glycolytic genes. The results suggest that Set9 epigenetically contributes to the glycolytic phenotype of cancer through HIF-1.

Citation Format: Qiong Liu, Hao Geng, Changhui Xue, Tomasz M. Beer, David Z. Qian. Regulation of HIF1a by Set9 lysine methyltransferase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 458. doi:10.1158/1538-7445.AM2014-458
Abstract 4588: Polylysine linker for successful manufacturing of hydrophobic peptides
Authors: Shakoori Firuz , Gangakhedkar Archana
Published: May 2015

Hydrophobic peptides are common to be used in cancer vaccinations. Hydrophobic peptides, continue to present challenges for large scale production in peptide industry. Experimental Procedures: Full sequence prepared by Solid phase Synthesis:The peptide was synthesized with Fmoc-Lys(Boc)-Wang resin by using standard Fmoc/tBu chemistry. Protecting groups used for amino acids are: tBu for Thr, Boc for Trp and Lys and Boc-Ser- OH was used as a hydrolysis-labile ester provider. Fmoc-Leu-OH was coupled on the side chain of Ser by DIC/DMAP (Amino acid/DIC/DMAP, 3/1.5/0.3 eq). The other amino acids were assembled by repetitive removal of the Fmoc protecting group by treating with 2% DBU/2% piperidine in DMF 30 minutes and coupling of protected amino acid by using DIC/HOBt/NMM (4:4:4 eq) in 180 minutes to overnight. Ninhydrin test was performed after each coupling to check the coupling efficiency. After the last coupling,resin was washed and dried, then treated with reagent K for cleavage and removal of the side chain protecting groups. Crude peptide was precipitated from cold ether and collected by filtration. Table.1

Summary of 4 synthesize results

Syn. NoLinker StructurePurity of CrudeDKP deletion: Product Hydrolysis YieldOverall Yield1No Linker61.4%12.3%2(O)-Ser56.5%1:252.8%12.5%3(O)-Ac-Ser40.8%1:370.6%28.2%4HMBA78.1%<1:2451.0%61.2%

Conclusion: American Peptide demonstrated the utility of a hydrophilic peptide ‘tail’ in purification of a hydrophobic peptide on both small (20-30 mmol) research scale and large (800 mmol) cGMP scales to provide the peptide in hundreds-of-grams quantities. The approach described allowed for both time-and cost-competitive manufacturing of this difficult peptide for clinical trials.

Citation Format: Firuz Shakoori, Archana Gangakhedkar. Polylysine linker for successful manufacturing of hydrophobic peptides. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2014-4588
Abstract 481: Dualistic regulation of NF-kB signaling by CD99 and EWS-FLI1 in Ewing sarcoma: impact on cell differentiation
Authors: Ventura Selena , Manzalini Chiara , Aryee Dave , Picci Piero , Kovar Heinrich , Scotlandi Katia
Published: May 2015

Introduction: Ewing sarcoma (ES) is a high-grade rare malignant bone tumor in children and adolescents. This disease is primarily characterized by: i) Chromosomal translocations that fuse EWS gene and a gene of the ETS family of transcription factors (around 85% EWS-FLI1 and 10% EWS-ERG, respectively) which are the oncogenic drivers of this tumor; ii) High expression of CD99, a cell surface glycoprotein virtually present in all of ES cells. EWS-FLI1 and CD99 were reported to have divergent roles in modulating ES malignancy and neural differentiation In this study we investigated the possible role of nuclear factor-kappa B (NF-kB) signaling pathway on the neural differentiation of ES cells and its relationship with EWS-FLI1 and CD99.

Materials and Methods: Transcriptional activity of several transcription factors (AP1, SRE, CREB, E2F and NF-kB) were evaluated by gene reporter assay. Using a knockdown approach in stable and transient conditions we silenced CD99, EWS-FLI1 and NF-kB itself, alone or in combination, in three ES cell lines (TC-71, IOR/CAR and ASP14). NF-kBp65 was also transiently overexpressed in TCshCD99 and CARshCD99 clones with pCMVp65 construct. Neural differentiation was detected evaluating β-III tubulin by immunofluorescence microscopy.

Results: NF-kB was found heterogeneously expressed in a panel of ES cell lines. Stable CD99 silencing was found to decrease NF-kB activity (pValue <0.05) without any differences at RNA and protein levels. On the contrary, EWS-FLI1 knockdown increased NF-kB transactivation (pValue <0.01). CD99 affects NF-kBp65 activity and its effect are dominant with respect to EWS-FLI1. Accordingly, EWS-FLI1 silencing was not able to revert the reduction of NF-kB activity observed after CD99 deprivation. In addition cells silenced for CD99 but not for EWS-FLI1 (TCshCD99 and CARshCD99 clones) showed a neural differentiated phenotype, which was lost when cells were induced to re-express NF-kB.

Conclusion: Our findings support the concept that inhibition of NF-kB is able to induce terminal neural differentiation of ES cells. This identifying NF-kB as a potential therapeutic target against Ewing sarcoma progression.

(Supported by AIRC_2013_IG 14049 and Liddy Shriver Foundation)

Citation Format: Selena Ventura, Chiara Manzalini, Dave Aryee, Piero Picci, Heinrich Kovar, Katia Scotlandi. Dualistic regulation of NF-kB signaling by CD99 and EWS-FLI1 in Ewing sarcoma: impact on cell differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 481. doi:10.1158/1538-7445.AM2014-481
Abstract 4811: Soluble biomarkers identify mCRC patients subgroups showing increased benefit from the VEGF signaling inhibitor cediranib in combination with chemotherapy
Authors: Pommier Aurelien J.C. , Spencer Stuart , Morgan Shethah , Womack Chris , Jurgensmeier Juliane M. , Critchlow Susan , Barry Simon T.
Published: May 2015

Identifying potential predictive biomarkers differentiating sensitivity/resistance to VEGF signaling inhibitors (VEGFi) will enhance use of these therapies. We used >580 pre and on-treatment serum samples and 259 tumours diagnostic biopsies from mCRC patients enrolled in a Phase III trial (HORIZON II) treated with chemotherapy ± cediranib(a VEGFi), to screen the potential predictive value of multiple serum proteins and tumour genes.

At baseline leptin was the most significant marker associated with OS of patients treated with chemo-ced (Pinteraction<0.001). Leptin concentration correlated with the body mass index (BMI) and obese patients (BMI>30) showed improved OS when treated with chemo-ced compared to non-obese patients (HR=0.55, 0.36-0.85, p=0.008). In the same patients, we identified two gene expression signatures associated with benefit on chemo-ced. Patients characterized by a high expression of GAHGL (Glucose metabolism, Angiogenesis, Hypoxia, Glutamine metabolism, Leptin signaling) (Pinteraction=0.028) and low expression of LOPS (Lipid metabolism, Oxidative activity, Proliferation and Serine synthesis) gene signature (Pinteraction=0.002) had improved OS in chemo-ced treated patients. Serum leptin concentration was found positively correlated with expression of genes involved in GAHGL in patients.

In addition a baseline signature (BS) of 47 biomarkers including VEGFA, VEGFD, VEGFR2, VEGFR3 and TIE-2, defined two distinct subgroups of patients. Patients treated with chemotherapy plus cediranib who had ‘high’ BS had shorter PFS (HR=1.82, p=0.003) than patients with ‘low’ BS. This BS had no effect on PFS of the patients treated with chemotherapy plus placebo.

Finally, we identified a profile of 16 pharmacodynamic (PD) proteins on treatment associated with PFS (HR=0.58, p<0.001) and OS (HR=0.52, p<0.001) in patients treated with chemotherapy plus cediranib. This PD profile had no effect on PFS and OS in patients treated with chemotherapy plus placebo.

This work provides original candidate biomarkers to select patients most likely to benefit from VEGFi therapies.

Citation Format: Aurelien J.C. Pommier, Stuart Spencer, Shethah Morgan, Chris Womack, Juliane M. Jurgensmeier, Susan Critchlow, Simon T. Barry. Soluble biomarkers identify mCRC patients subgroups showing increased benefit from the VEGF signaling inhibitor cediranib in combination with chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4811. doi:10.1158/1538-7445.AM2014-4811
Abstract 4843: Neutrophil elastase enhances adaptive immunity via increase of peptide presentation and downregulation of T cell inhibitory molecules
Authors: Chawla Akhil , Philips Anne V. , Qiao Na , Sukhumalchandra Pariya , Kerros Celine , Alatrash Gheath , Molldrem Jeffrey J. , Mittendorf Elizabeth A.
Published: May 2015

Introduction: Neutrophil elastase (NE) is an inflammatory mediator that is taken up by breast cancer cells. We have previously shown that NE uptake increases susceptibility to lysis by cytotoxic T lymphocytes (CTL) targeting the tumor antigens PR1 and cyclin E. We investigated whether NE uptake alters adaptive immunity by affecting MHC class I antigen presentation and T cell inhibitory molecules.

Methods: MDA-MB-231 breast cancer cells were maintained in standard media ± NE. Healthy donor HLA-A2+ peripheral blood mononuclear cells were used to generate antigen specific CTL by pulsing dendritic cells with E75, a HER2-derived epitope we are currently investigating as a vaccine in clinical trials. Calcein-AM cytotoxicity assays evaluated E75-specific lysis. Flow cytometry was used to show NE uptake, HLA-A2, pan-HLA class I (HLA-ABC) and PD-L1 surface expression. Western blot analysis was used to show total MHC class I heavy chain, B2M, LMP2, TAP1, calnexin and tapasin expression. Relative PD-L1 mRNA levels were determined using qPCR.

Results: NE uptake enhanced tumor cell lysis by E75-specific CTL at all effector to target (E:T) ratios (lysis for 20 to 1 E:T ratio was 50% ± 7 for NE treated vs 28% ± 7 for untreated, p < 0.05), and led to an increase in HLA-A2 and HLA-ABC surface expression (p < 0.05; Table). However, NE uptake did not change total expression of MHC class I or components of the MHC class I pathway. Additionally, NE uptake led to a decrease in PD-L1 surface expression and transcript levels (p < 0.05; Table).

Conclusion: NE increases tumor antigen presentation as well as MHC class I on the cell surface. However, NE uptake does not change total MHC class I expression or expression of MHC class I antigen processing components, suggesting that NE increases peptide availability as a mechanism of increased MHC class I antigen presentation. NE also leads to the downregulation of PD-L1. Together, our data indicate that NE uptake enhances adaptive immune responses.

HLA-A2 Surface Expression (MFI)HLA-ABC Surface Expression (MFI)PD-L1 Surface Expression (MFI)PD-L1 mRNA Expression (RQ)NE Treated (Mean ± SD)7341 ± 8671317 ± 1201723 ± 550.274 ± 0.02Untreated (Mean ± SD)5144 ± 272802 ± 1336966 ± 4511

Citation Format: Akhil Chawla, Anne V. Philips, Na Qiao, Pariya Sukhumalchandra, Celine Kerros, Gheath Alatrash, Jeffrey J. Molldrem, Elizabeth A. Mittendorf. Neutrophil elastase enhances adaptive immunity via increase of peptide presentation and downregulation of T cell inhibitory molecules. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4843. doi:10.1158/1538-7445.AM2014-4843
Abstract 4847: Alcoholic chronic pancreatitis increases tissue transformed macrophages (M2) which promote pancreatic cancer lesions in a Kras mouse model
Authors: Edderkaoui Mouad , Grippo Paul , Benhaddou Hajar , Ouhaddi Yassine , Tsukamoto Hidekazu , Swartwood Steven , Knudsen Beatrice , Goodridge Helen S. , Habtezion Aida , Go Vay-Liang , Pandol Stephen J.
Published: May 2015

Background: Alcoholic chronic pancreatitis (ACP) is a major risk factor for pancreatic cancer; however, the mechanism through which it promotes cancer is not known. In this study we developed a mouse model of pancreatic cancer induced by ACP and analyzed the effect of the interaction between macrophages and pancreatic cancer cells on progression of the disease.

Methods: Pdx1-Cre;LSL-Kras transgenic and wild-type (WT) mice were fed Lieber DeCarli control or alcohol diet for 6 weeks. Mice were subjected to 7-hourly cerulein injections (50µg/kg) twice a week for the last 3 weeks of the diet to induce chronic pancreatitis. Pancreatic intraepithelial neoplastic (PanIN) lesions, proliferation, fibrosis, inflammation, EMT and stem cell markers were analyzed by immunohistochemistry and Western. Monocytes/ macrophages from mice and their conditioned medium were applied to the pancreatic cancer cell line MIA PaCa-2. Cell survival and apoptosis were measured by MTT and ELISA. Expression of cytokines/ chemokines and growth factors by cancer cells and macrophages was measured by multiplex assay analysis.

Results: ACP increased fibrosis, stellate cell activation and inflammation in both WT and transgenic mouse pancreata. The increases were significantly greater in the transgenic mice. Further, ACP increased the number and stage of PanIN lesions in the pancreata of transgenic mice. EMT markers were stimulated by ACP in both WT and transgenic mice. Markers of type 2 macrophages (M2) were greater in ACP model compared to control mice. M2 macrophages conditioned media stimulated proliferation, EMT and cancer stemness in cancer cells. Cancer cells or PanIN cells conditioned media stimulated transformation of macrophages to the type 2 phenotype.

Conclusion: The results indicate that ACP promotes pancreatic cancer progression in transgenic mice. Our data suggest ACP as well as PanIN and cancer cells stimulate the phenotype change of macrophages toward type 2, which then stimulates cancer cell proliferation, EMT and cancer stemness. These results suggest that tissue macrophages play a key role in the increased risk for pancreatic cancer in patients with alcoholic pancreatitis.

Citation Format: Mouad Edderkaoui, Paul Grippo, Hajar Benhaddou, Yassine Ouhaddi, Hidekazu Tsukamoto, Steven Swartwood, Beatrice Knudsen, Helen S. Goodridge, Aida Habtezion, Vay-Liang Go, Stephen J. Pandol. Alcoholic chronic pancreatitis increases tissue transformed macrophages (M2) which promote pancreatic cancer lesions in a Kras mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4847. doi:10.1158/1538-7445.AM2014-4847
Abstract 4851: Kinase domain activation of FGFR2 yields high-grade lung adenocarcinoma sensitive to a pan-FGFR inhibitor in a mouse model of NSCLC
Authors: Akbay Esra A. , Tchaicha Jeremy H. , Altabef Abigail , Mikse Oliver R. , Kikuchi Eiki , Rhee Kevin , Liao Rachel , Bronson Roderick T. , Sholl Lynette M. , Meyerson Matthew , Hammerman Peter S. , Wong Kwok-Kin
Published: May 2015

Somatic mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) have been found in 4-5% of patients diagnosed with non-small cell lung cancer (NSCLC). FGFR2 and other FGFR kinase family gene alterations have been found in lung squamous cell carcinoma, adenocarcinoma, and other malignancies though mouse models of FGFR driven lung cancers have not been reported. Here, we generated a genetically engineered mouse model (GEMM) of NSCLC driven by a kinase domain mutation in FGFR2. Combined with p53 ablation, primary grade III/IV adenocarcinoma was induced in the lung epithelial compartment exhibiting locally invasive and pleiotropic tendencies largely made up of multinucleated cells. Tumors were acutely sensitive to pharmacological inhibition of FGFR signaling. In preliminary studies tumors also responded to Programmed death pathway immune checkpoint blockade using anti-PD-1 antibody arguing for activated immune evasion mechanisms in this model.This is the first autochthonous FGFR2-driven lung cancer GEMM that can be applied across different cancer indications in a preclinical setting.

Citation Format: Esra A. Akbay, Jeremy H. Tchaicha, Abigail Altabef, Oliver R. Mikse, Eiki Kikuchi, Kevin Rhee, Rachel Liao, Roderick T. Bronson, Lynette M. Sholl, Matthew Meyerson, Peter S. Hammerman, Kwok-Kin Wong. Kinase domain activation of FGFR2 yields high-grade lung adenocarcinoma sensitive to a pan-FGFR inhibitor in a mouse model of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2014-4851
Abstract 4864: Tumor promoting role of NF-kappa B in the glioma environment
Authors: Kwiatkowska Aneta , Nandhu Mohan Sobana , Viapiano Mariano Sebastian
Published: May 2015

Introduction: Gliomas are the most common primary malignant brain tumors. Their characteristic features are extensive growth and lack of the clear borders due to diffuse invasion of surrounding tissue. Invasive glioma cells have been shown to interact through soluble signals with other cells in the neural microenvironment, which is normally highly inhibitory to cell motility. This communication changes the behavior of the tumor microenvironment to become permissive and facilitate tumor invasion. Here, we investigated the role of NFκB signaling in the glioma microenvironment and demonstrate that activation of NFκB in neural cells is critical for tumor growth and invasion. Methodology: We analyzed the growth and invasion of malignant gliomas using an NFκB-deficient mouse model (RelA(p65)-/- TNFR1-/-). Since p65-/- mice do not survive to adulthood, mouse glioma cells were implanted in p65+/- mice (which have significantly reduced NFκB activity) or their control littermates. Organotypic cultures of p65-/- brain slices and cultures of p65-/- neonatal astrocytes were used to assess the role of active NFκB signaling in astroglia on tumor invasion. Results: Using the mouse glioma cell line GL261 (poorly invasive) we observed significantly reduced tumor volume in NFκB -deficient mice compared to wild-type littermates. Further studies using mouse glioma stem-like cells Mut4 (highly invasive; GFAP-Cre: Nf1loxP/+ Trp53-/+ PTENloxP/+; Alcantara-Llaguno et al., Cancer Cell, 2009) demonstrated reduction both in tumor size and extent of invasion in the NFκB-deficient microenvironment. Importantly, immunohistochemical analysis demonstrated significantly reduced number of protoplasmic GFAP- positive astrocytes surrounding the tumors in p65+/- brains. Further analysis in brain slice cultures showed that invasion of glioblastoma cells was highly reduced through p65+/- tissue. Moreover, p65+/- astrocytes exhibited significantly reduced responses towards glioma cells, including reduced invasion through Matrigel in response to signals released by

GL261 cells. Conclusions: Our results strongly suggest that NFκB signaling may be involved in the recruitment or activation of reactive astrocytes surrounding the invasive borders of gliomas. This activation is likely necessary for enhanced tumor invasion through brain tissue. Our results highlight the relevance of the astroglial compartment as a collateral target that should be considered in future strategies targeting brain tumor invasion.

Citation Format: Aneta Kwiatkowska, Mohan Sobana Nandhu, Mariano Sebastian Viapiano. Tumor promoting role of NF-kappa B in the glioma environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4864. doi:10.1158/1538-7445.AM2014-4864
Abstract 4980: Inhibiting carbonic anhydrase IX targets hypoxic small cell lung cancer cells
Authors: Bryant Jennifer L. , Meredith Suzanne L. , Gieling Roben G. , Telfer Brian A. , Babur Muhammad , Supuran Claudiu T. , Williams Kaye J. , White Anne
Published: May 2015

Small-cell lung cancer (SCLC) is an extremely aggressive disease, exhibiting acquired resistance to therapy and poor patient prognosis. Therapy resistance can in part be attributed to tumor hypoxia. Hypoxic SCLC cells up-regulate carbonic anhydrase IX (CA IX) in order to survive. CA IX is involved in pH maintenance of cells and presents a novel therapeutic target for SCLC. This study aimed to investigate the potential of inhibiting CA IX in SCLC as a novel therapeutic target.

Hypoxic conditions (0.1% O2) increased CA IX expression over 24 hours in both COR-L24 and DMS 79 SCLC cells in culture. S4 belongs to a sulfamate class of carbonic anhydrase inhibitors and has shown anti-metastatic properties in breast cancer xenografts. Treatment with 30μM and 100μM S4 significantly decreased CA IX protein levels in hypoxic COR-L24 and DMS 79 SCLC cells, respectively (p<0.05 for both treatments). Exposure to S4 for 24 hours resulted in predominantly hypoxic cell death in both cell lines (p<0.05). Combining S4 with etoposide (0.3µM) and cisplatin (0.5µM) chemotherapeutic agents resulted in additional cell death, which was also seen under normoxic conditions (20% O2). Sub-cutaneous xenograft tumors were grown in nude mice using either COR-L24 or DMS 79 SCLC cells. Resultant tumors (1000mm3) expressed CA IX largely adjacent to regions of necrosis. Treating mice with 50mg/kg S4 on two consecutive days each week resulted in greater necrosis than saline controls, coupled with up-regulation of CA IX. Analyzing tumors for characteristic neuroendocrine (pro-opiomelanocortin) and epithelial (cytokeratin) markers revealed heterogeneous expression; however, this did not correlate with CA IX expression and is not altered in response to treatment.

In summary, CA IX is upregulated in hypoxic SCLC cells and tumors. Inhibition of CA IX resulted in hypoxic cell death and had an additive effect to the response observed with chemotherapy treatment alone. This suggests that directly targeting hypoxic SCLC cells via CA IX inhibition holds promise in improving treatment outcome for this aggressive disease.

Citation Format: Jennifer L. Bryant, Suzanne L. Meredith, Roben G. Gieling, Brian A. Telfer, Muhammad Babur, Claudiu T. Supuran, Kaye J. Williams, Anne White. Inhibiting carbonic anhydrase IX targets hypoxic small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4980. doi:10.1158/1538-7445.AM2014-4980
Abstract 5063: PSCA as a potential therapeutic and prognostic biomarker for common cancer
Authors: Matsuda Koichi , Tanikawa Chizu , Nakamura Yusuke
Published: May 2015

SNP rs2294008 in PSCA was shown to be associated with bladder and gastric cancer risk among European and Asian. PSCA was upregulated in various cancer tissues, and PSCA expression level was associated with tumor stage and poor prognosis. Thus PSCA is consider as promising therapeutic target of cancers. Our group revealed that T allele of rs2294008 encodes a translation initiation codon upstream of the reported site and changes protein localization from the cytoplasm to the cell surface. To further investigate the role of this variation in Japanese population, we genotyped using bladder and lung cancer samples. As a result, we found the significant association with bladder and lung cancer risk (OR=1.20 and 1.09 with P-value of 0.0092 and 0.0126). In addition, we found that T allele was associated with higher level of mRNA expression and T type of PSCA protein was secreted into the culture medium. These finding suggest that PSCA genotype and serum PSCA protein would be good biomarkers of disease risk, prognosis, and response to treatment using anti-PSCA antibody, implicating the clinical usefulness of genomic analysis for the treatment of common cancer.

Citation Format: Koichi Matsuda, Chizu Tanikawa, Yusuke Nakamura. PSCA as a potential therapeutic and prognostic biomarker for common cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5063. doi:10.1158/1538-7445.AM2014-5063
Abstract 5151: A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hyper-trimethylation
Authors: Ott Heidi , Graves Alan , Pappalardi Melissa , Kruger Ryan , Tummino Peter , Creasy Caretha , McCabe Michael T.
Published: May 2015

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin's lymphoma. While the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates di-methylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a mono-methylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants which dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell derived cancer cell lines identified a cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641 or A677 mutant lines. Treatment of A687V EZH2 mutant cells with GSK126, a selective EZH2 inhibitor, induced global H3K27me3 demethylation, robust gene activation, caspase activation, and decreased proliferation. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival.

Citation Format: Heidi Ott, Alan Graves, Melissa Pappalardi, Ryan Kruger, Peter Tummino, Caretha Creasy, Michael T. McCabe. A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hyper-trimethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5151. doi:10.1158/1538-7445.AM2014-5151
Abstract 5205: CCND1 is a miR-193b target in prostate cancer
Authors: Tuppurainen Kirsi M. , Rauhala Hanna E. , Scaravilli Mauro , Vessella Robert L. , Visakorpi Tapio
Published: May 2015

Prostate cancer is the most frequent cancer of men in Western countries and causes second most of the cancer-related deaths. In addition to genetic changes also epigenetic alterations and micro-RNAs (miRNAs) are important in prostate cancer development. Our group has previously shown that miR-193b is hypermethylated in 22Rv1 prostate cancer cell line and that it is putative tumor suppressor in prostate cancer. In melanoma and hepatocellular carcinoma, it has been shown that CCND1, encoding cyclin D1 protein, is among the target genes of miR-193b. The aim of this project was to study if CCND1 is a target of miR-193b in prostate cancer.

According to qRT-PCR and microarray analyses, the expression of CCND1 and miR-193b were inversely correlated in prostate cancer cell lines (PC-3, VCaP, 22Rv1, LAPC4, DU145, LNCaP) and in LuCaP xenografts. Using a luciferase reporter gene assay in 22Rv1 cells, we showed that miR-193b targets CCND1 3′UTR. In 22Rv1 cells with low endogenous miR-193b expression, transient pre-miR-193b transfection reduced CCND1 mRNA levels and cyclin D1 and phospho-RB protein levels. By rescue experiment we confirmed that the downregulation of cyclin D1 and phospho-RB proteins is due to miR-193b. When prostate cancer cell lines were treated with cdk4/6 inhibitor, 22Rv1 and VCaP cells with low miR-193b and high CCND1 expression showed significant growth retardation. In contrast, the inhibitor did not have any effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Furthermore, immunohistochemical analysis of 337 specimens showed that cyclin D1 is expressed significantly higher in castration resistant compared to hormone-naïve prostate cancers. Therefore, we conclude that CCND1 is one of the miR-193b targets in prostate cancer.

Citation Format: Kirsi M. Tuppurainen, Hanna E. Rauhala, Mauro Scaravilli, Robert L. Vessella, Tapio Visakorpi. CCND1 is a miR-193b target in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2014-5205
Abstract 5268: The expression of lemur tyrosine kinase-3 in high grade pediatric tumors
Authors: Mazarakis Nektarios K. , Smith Stuart , Vraka Aikaterini , Macarthur Donald , Grundy Richard , Stebbing Justin , Giamas George
Published: May 2015

High grade gliomas are the most common primary brain tumors in adults, but also occur in childhood, with a poor prognosis in all age groups . Lemur tyrosine kinase 3 (LMTK3) is a transmembrane tyrosine kinase that seems to play a pivotal role in oncogenesis in breast cancer. It regulates the estrogen receptor alpha and is evolutionarily selected for in humans compared to non-human primates. LMTK3 expression levels were strongly associated with survival and therapy response.This study assesses the role of LMTK3 in pediatric high grade brain tumors at both the protein and mRNA levels.

We initially assessed by immunohistochemistry (IHC) the expression levels of LMTK3 in well described cohorts of patient specimens with brain cancer. First, we performed an IHC analysis using a brain tumor tissue microarray (TMA) purchased from US Biomax (#T175), in order to examine the LMTK3 levels in a variety of brain tumors.

Preliminary analysis revealed variable LMTK3 expression levels in brain tissue, scored as negative, low, moderate and high. We demonstrate that LMTK3 appears to be expressed at low levels at normal brain tissue samples and epithelial meningioma tissues (benign tumors). On the contrary, LMTK3 was expressed at different levels in cases with grade 2 or grade 4 gliomas. Further immunohistochemical analysis was therefore undertaken on a selection of TMAs containing pediatric high grade gliomas. Genome wide gene expression analysis (using the Affymetrix U133plus2 chip) previously undertaken on a cohort of pediatric high grade gliomas demonstrated measurable LMTK3 mRNA expression in many samples. Our preliminary analysis was correlated with various tumor characteristics that are available in Nottingham datasets (including demographic and overall survival data). In conclusion, LMTK3 is expressed in pediatric high grade gliomas and may have differential expression depending on the histological grade of the tumor. This work will lead to larger scale studies to elucidate the exact role of LMTK3 in high grade brain neoplasms.

Citation Format: Nektarios K. Mazarakis, Stuart Smith, Aikaterini Vraka, Donald Macarthur, Richard Grundy, Justin Stebbing, George Giamas. The expression of lemur tyrosine kinase-3 in high grade pediatric tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2014-5268
Abstract 5271: Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer
Authors: Togashi Yosuke , Sakamoto Hiroki , Hayashi Hidetoshi , Terashima Masato , Velasco Marco A. de , Fujita Yoshihiko , Kodera Yasuo , Sakai Kazuko , Tomida Shuta , Kitano Masayuki , Kudo Masatoshi , Nishio Kazuto
Published: May 2015

Background: Transforming growth factor, beta (TGFB) signal is considered to be a tumor suppressive pathway based on the frequent genomic deletion of the SMAD4 gene in pancreatic cancer (PC); however; the role of the activin signal, which also belongs to the TGFB superfamily, remains largely unclear.

Methods and Results: We found a homozygous deletion of the activin A receptor, type IB (ACVR1B) gene in 2 out of 8 PC cell lines using array-comparative genomic hybridization, and the absence of ACVR1B mRNA and protein expression was confirmed in these 2 cell lines. Activin A stimulation inhibited cellular growth and increased the phosphorylation level of SMAD2 and the expression level of p21CIP1/WAF1 in the Sui66 cell line (wild-type ACVR1B and SMAD4 genes) but not in the Sui68 cell line (homozygous deletion of ACVR1B gene). Stable ACVR1B-knockdown using short hairpin RNA cancelled the effects of activin A on the cellular growth of the PC cell lines. In addition, ACVR1B-knockdown significantly enhanced the colony formation abilities and cellular growth, compared with controls. In a xenograft study, ACVR1B-knockdown resulted in a significantly elevated level of tumorigenesis and a larger tumor volume, compared with the control. Furthermore, in clinical samples, 6 of the 29 PC samples (20.7%) carried a deletion of the ACVR1B gene, while 10 of the 29 samples (34.5%) carried a deletion of the SMAD4 gene. Of note, 5 of the 6 samples with a deletion of the ACVR1B gene also had a deletion of the SMAD4 gene.

Conclusion: We identified a homozygous deletion of the ACVR1B gene in PC cell lines and clinical samples and proposed that the deletion of the ACVR1B gene may mediate an aggressive cancer phenotype in PC. Our findings provide novel insight into the role of the activin signal in PC.

Citation Format: Yosuke Togashi, Hiroki Sakamoto, Hidetoshi Hayashi, Masato Terashima, Marco A. de Velasco, Yoshihiko Fujita, Yasuo Kodera, Kazuko Sakai, Shuta Tomida, Masayuki Kitano, Masatoshi Kudo, Kazuto Nishio. Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5271. doi:10.1158/1538-7445.AM2014-5271
Abstract 5388: High throughput toxicity assay for three-dimensional cell cultures
Authors: Trumpi Kari , Egan David A. , Vellinga Thomas T. , Rinkes Inne H.M Borel , Kranenburg Onno W.
Published: May 2015

Introduction:

Three-dimensional cell culture methods are currently not widely used for cancer drug discovery due to the specific imaging demands of the systems. In particular there is a need for well validated robust high throughput methods for image-based assays that can be used for high content screening.

Methods:

Therefore we have developed an image-based three-dimensional high throughput toxicity assay. We used two platforms, the Thermo Scientific ArrayScan VTi and BD FACSCanto™, to characterize antiproliferative potency, cell death and cell cycle distribution. Cultures of uniform spheroids were treated with Irinotecan either alone or in combination with the ABC transporter inhibitor Valspodar, which blocks irinotecan efflux. All drugs were dispensed using a Tecan HP-D300 digital dispenser. Treatment-induced effects were analyzed after 48, 72 or 96 hours with both platforms using dihydro derivatives of fluorescent live cell dyes such as dihydrocalcein, AM and DNA-binding dyes such as DRAQ5™ and Propidium Iodide.

Results:

All cell lines tested on the two platforms showed the expected effects of Irinotecan alone or in combination with Valspodar and correlations between the Thermo Scientific ArrayScan VTi and BD FACSCanto™ were high, with Pearson tests varying from 0.76 to 0.95, p < 0.05, depending on the cell line used. All results were reproducible and showed minor variation.

Conclusion:

The demonstrated approach provides an easy-to-handle, low-cost, accurate and reproducible high content method for measuring drug efficacy in three-dimensional cell cultures. This method can be used for high throughput drug discovery screens and personalized cancer care.

Citation Format: Kari Trumpi, David A. Egan, Thomas T. Vellinga, Inne H.M Borel Rinkes, Onno W. Kranenburg. High throughput toxicity assay for three-dimensional cell cultures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5388. doi:10.1158/1538-7445.AM2014-5388
Abstract 5439: Metabolomic study of EGFR drug resistance mechanisms
Authors: Zhao Yang , Tan Bo , Kuo Ming-Shang , Liu Ling , Breyer Matthew D.
Published: May 2015

Therapies targeting EGFR often fail after certain period of treatment due to development of resistance. There are many causes of resistance such as genetic mutations of KRAS, BRAF, etc.. Overexpression of other receptor tyrosine kinases can also lead to resistance of EGFR therapies. A therapeutic targeting metabolic phenotypes that can overcome multiple resistance mechanisms is highly desired. In order to identify the Achilles heel of resistant cell metabolism, we investigated the metabolic effects of the current EGFR targeting agents as well as the metabolic differences between sensitive and resistant cancer cells.

Citation Format: Yang Zhao, Bo Tan, Ming-Shang Kuo, Ling Liu, Matthew D. Breyer. Metabolomic study of EGFR drug resistance mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5439. doi:10.1158/1538-7445.AM2014-5439
Abstract 5474: The formin, DIAPH3, regulates response to MT stabilizing drugs in prostate and breast cancer
Authors: Morley Samantha , Pollan Sara G. , You Sungyong , Knudsen Beatrice S. , Freeman Michael
Published: May 2015

Diaphanous-related formin-3 (DIAPH3), a cytoskeletal regulator of the formin family, is lost at high frequency in metastatic breast and prostate cancers and has characteristics of a non-canonical metastasis suppressor. We previously showed that DIAPH3 silencing evokes a transition to an “amoeboid” phenotype, characterized by rapid motility, RhoA/ROCK and ERK pathway activation, dynamic membrane blebbing and increased shedding of microvesicles. Here we demonstrate that DIAPH3 loss reduces microtubule (MT) stability in prostate and breast cancer cell lines, and increases the cells' sensitivity to the taxanes, paclitaxel and docetaxel and the non-taxane, epothilone B. DIAPH3 silencing reduces membrane localization of the multidrug resistance-associated protein, MRP1, a regulator of taxane efflux, and increases intracellular accumulation of fluorescent paclitaxel. Baseline acetylation of tubulin, which is proportional to the number of stable MT, is lower in DIAPH3-silenced cells. In contrast, the fold-increase of tubulin acetylation after treatment with MT stabilizing drugs is greater in DIAPH3 knockdown compared to control cells. Further, cytotoxicity induced by paclitaxel, docetaxel or epothilone B is enhanced by DIAPH3 silencing. Analysis of gene expression profiles of breast cancer patients treated with chemotherapeutic regimens containing taxanes in a neoadjuvant phase-2 clinical trial revealed that low DIAPH3 expression is associated with increased relapse free survival. Collectively, the data demonstrate that DIAPH3 deficiency increases responsiveness to widely used MT stabilizing drugs. These findings have implications for the inclusion of DIAPH3 in biomarker panels that stratify patients for treatment with taxanes, in particular in the neoadjuvant setting.

Citation Format: Samantha Morley, Sara G. Pollan, Sungyong You, Beatrice S. Knudsen, Michael Freeman. The formin, DIAPH3, regulates response to MT stabilizing drugs in prostate and breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2014-5474