bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3877: ABT-737 synergizes with bortezomib to induce apoptosis: Activation of Bid, Bak, and Bax in the apoptotic response in malignant human glioma cells
Authors: Jane Esther P. , Premkumar Daniel , DiDomenico Joseph , Agostino Naomi , Vukmer Natalie , Pollack Ian
Published: August 2012

Glioblastomas are invasive tumors with poor prognosis despite current therapies. ABT-737 has monotherapeutic toxicity against leukemia, lymphoma and other malignancies, and we and others have noted some antiglioma activity, although insufficient to warrant use as monotherapy. We have recently demonstrated that proteasome inhibitors, such as bortezomib, dramatically sensitized highly resistant glioma cells to apoptosis induction, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to ABT-737 inhibition in glioma cells. Although primary cells from glioblastoma multiforme (GBM) patients and established glioma cell lines did not show significant induction of apoptosis with ABT-737 treatment alone, the combination of ABT-737 plus bortezomib, significantly enhanced apoptosis. The enhanced efficacy was due to proapoptotic mitochondrial injury and increased generation of reactive oxygen species. Our results also revealed that combination of bortezomib/ABT-73 enhanced apoptosis by increasing Bid cleavage, Noxa upregulation, Bak, Bax activation, and cytochrome c release. Further downregulation of Mcl-1 using shRNA enhanced cell killing by the bortezomib/ABT-737 combination. The significantly enhanced antitumor activity that results from the combination of bortezomib and ABT-737 offers promise as a novel treatment for glioma patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3877. doi:1538-7445.AM2012-3877
Abstract 3879: High-throughput, homogeneous in cyto assays to monitor histone H3 post-translational modifications
Authors: Beaudet Lucille , Sergerie Jean-Philippe Lévesque , Boulé Marie , Labonté Anne , Michaud Jean-François , Rouleau Nathalie , Arcand Mathieu
Published: August 2012

Histone proteins are an integral part of DNA packaging into chromatin, a dynamic process partly regulated by post-translation modification of histone N-terminal tails. Aberrant histone acetylation (ac) or methylation (me) levels is associated with a number of diseases. For example, histone deacetylases (HDACs and sirtuins) regulate the expression of many genes involved in neurodegeneration and can be aberrantly expressed in different tumors. In addition, deregulation of the methyl marks H3K4me3 and H3K27me3 are found associated with the development of several types of cancer. This work presents the development of high-throughput AlphaLISA® in cyto assays to monitor four specific histone marks: H3K9ac, H3K4me2, H3K27ac and H3K27me3. These assays were performed using an all-in-one well histone extraction protocol requiring no acid extraction or centrifugation steps. The level of each histone mark was first measured in cell titration experiments, seeding cells from 500 to 10 000 in 384 well plates. The chemical modulation of H3K4me2, H3K9ac and H3K27ac was monitored following overnight treatment of HeLa, HEK293, and Jurkat cells with the non-selective histone deacetylase inhibitors sodium butyrate and trichostatin A. Signal increases were all corroborated by Western blot analysis using the same antibodies as in the AlphaLISA detection assay. In the absence of chemical modulation, measurement of different H3K27me3 mark levels was performed in two B cell lymphoma cell lines: OCI-LY-19 and SU-DHL-6. OCI-LY-19 cells express wild-type EZH2 methyltransferase while SU-DHL-6 cells bear a heterozygous EZH2 mutation (Y641N) that alters its substrate selectivity, resulting in increased H3K27me3 levels. These novel cell-based assays showed suitability for high-throughput screening (HTS) protocols as demonstrated by Z’ factors superior to 0.6 for all four marks. In summary, these homogeneous cell-based AlphaLISA assays allow the simple and rapid monitoring of cellular histone H3 mark levels.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3879. doi:1538-7445.AM2012-3879
Abstract 3880: Application of tyramide signal amplification technology to high content analysis of cellular kinase translocation assays
Authors: Hurt Stephen , Campisano Michael , Letzsch Stefan
Published: August 2012

Deregulation of cellular kinase signaling pathways plays a key role in the ability of cancer cells to sustain uncontrolled proliferation, resist apoptosis, and undergo metastasis. The activation of kinase transduction pathways commonly involves the translocation of various molecular components of the pathway from one subcellular compartment to another. For example, growth factor stimulation of cells activates signaling pathways that are regulated by the level of phosphorylation of protein kinases such as protein kinase C (PKC) and protein kinase B (PKB/Akt); PKC and Akt are first recruited from the cytosol to the cell membrane where the phosphorylation event occurs. High Content Analysis (HCA) assays have become important tools in screening for potential new drugs to modulate aberrant signaling pathways. Confocal imaging provides the highest degree of spatial resolution, and can also yield lower background signals than wide-field imaging. The downside of confocal imaging is that it requires longer signal acquisition times, and it may be difficult to detect targets present in low abundance. The longer acquisition time can also result in decreased throughput when HCA is used as the detection mode for high throughput screening. Tyramide signal amplification (TSA) is an enzyme-based technology that has previous been used to enhance signal sensitivity in immunochemical staining procedures. We have applied TSA technology to the HCA of cellular kinase translocation assays using the Operetta High Content Imaging System in confocal fluorescence mode. HeLa cells were treated with compounds that induced kinase cytosol to plasma membrane translocation. Kinase localization was measured using a texture-based image analysis algorithm. We compared the signal intensity with and without TSA amplification. We will present data showing that TSA enhanced the signal intensity by at least an order of magnitude without a loss of resolution. As a result, the detection antibody can be reduced 5-fold, and the exposure time reduced 10-fold. In conclusion, TSA amplification of HCA kinase translocation assays can significantly increase sample throughput when screening compound libraries.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3880. doi:1538-7445.AM2012-3880
Abstract 3881: BacMam-enabled biochemical and cellular assays to assess inhibitors of full-length receptor tyrosine kinases
Authors: Hallis Tina , Lebakken Connie , Reichling Laurie , Ellefson Jason , Carlson Coby , Hermanson Spencer , Bi Kun , Riddle Steve
Published: August 2012

Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881
Abstract 3887: A screen for new cancer specific drugs that target centrosome clustering
Authors: Kawamura Eiko , Fielding Andrew , Kannan Nagarajan , Eaves Connie , Roberge Michel , Dedhar Shoukat
Published: August 2012

Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain extra centrosomes. Such cancer cells then achieve bipolar division by clustering centrosomes into two functional poles. Our lab has previously shown that inhibition of Integrin-linked kinase, which is a signaling and scaffold protein in focal adhesion and is also a centrosomal protein, results in scattered centrosomes and multipolar spindles, which leads to cancer specific cell death (Fielding et al., 2011, 30:521-534, Oncogene). A major problem with clinically used anti-mitotic drugs, such as taxol, is their toxicity in normal cells. Our goal is to develop new drugs that act specifically on cancer cells through targeting extra centrosomes without affecting normal cells. To discover new drugs, we established a high-content screen that automatically detects cells with de-clustered centrosomes. BT-549, a breast cancer cell line, was used for the screen, since it is known to effectively cluster centrosomes. The Canadian Chemical Biology Network enabled us to test chemical libraries predicted to have drug-like properties. For screening, cells were grown in 96 well plates, treated with test compounds for 5 hours, and subjected to immunofluorescence to examine centrosome arrangement in mitotic cells. Images were taken and analyzed with an automated fluorescence imager, Cellomics Array Scan VTI. We have thus screened over 6000 compounds from which we identified 18 hits. Fifteen of these were confirmed to inhibit centrosome clustering in the cells cultured under standard conditions, and induce an arrest in mitosis. Three compounds are structurally similar, suggesting a common structural motif as preventing centrosome clustering. To compare the effects of these drugs on normal and cancer cells, the viability of several breast and other cancer cell lines, an immortalized model of normal human mammary epithelial cells (MCF10A), and freshly isolated primary normal human mammary epithelial cells were examined after two days of drug treatment using the MTT assay. These comparisons identified some compounds that selectively reduced the viability of cancer cells, but not normal mammary epithelial cells suggesting their potential for cancer-specific therapy. These small molecules are currently being tested in orthotopic and xenograft models, and will be studied for target identification and mechanism of action for further drug development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3887. doi:1538-7445.AM2012-3887
Abstract 3894: Discovery, synthesis and SAR studies of a novel nonpeptidic reversible proteosome inhibitor with low nano-molar chymotrypsin like activity
Authors: Ozcan Sevil , Kazi Aslamuzzaman , Sun Nan N. , Daniel Kenyon , Guida Wayne C. , Lawrence Harshani R. , Sebti Said M.
Published: August 2012

The development of proteosome inhibitors for CT-L activity has been the subject of considerable interest in the treatment of cancer due to its critical role in maintaining homeostasis and hence its key position in many cellular processes. Proteosome inhibitors are classified as reversible or irreversible inhibitors according to their chemical structure and their mechanism of inhibition. Irreversible inhibitors possess a chemically reactive group, such as aldehydes, boronic acids, epoxy ketones that react with catalytic Thr1-O covalently, where as reversible inhibitors inhibit proteosome non-covalently via H-bond interactions (electrostatic and/or van der Waals). Bortezomib, a peptide boronic acid analog, is the first clinically approved proteosome inhibitor and is found to be an irreversible or slow reversible inhibitor that forms covalent bond with the N-terminal nucleophilic Thr1 in the β5 subunit of the proteasome. Reversible proteosome inhibitors (peptidic molecules) reported to date inhibit the proteosome non-covalently. Similar to Bortezomib, MLN9708 is a modified dipeptidyl boronic acid, which hydroylses immediately in plasma to MLN2238, is a potent, reversible and specific inhibitor of the chymotrypsin-like subunit of the proteasome. Small molecules as reversible proteosome inhibitors are less extensively investigated. In this study, we present the discovery of PI-184, non-peptidic small molecule with a pyridine and oxadiazole pharmacore as a reversible, potent and a novel proteasome inhibitor. Structure Activity relationship (SAR) guided synthesis of analogs around PI-184 led to the discovery of highly potent compounds with in-vivo chymotrypsin-like inhibitory activity. Here we present the detailed SAR, in-vitro and in-vivo proteasome activity of PI-184 class of compounds as potential anti-cancer agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3894. doi:1538-7445.AM2012-3894
Abstract 3896: New heat shock protein 90 inhibitors based on the 1,2,3-triazole structure
Authors: Giannini Giuseppe , Taddei Maurizio , Ferrini Serena , Alloatti Domenico , Brunetti Tiziana , Vincenti Silvia , Vesci Loredana , Castorina Massimo , Milazzo Ferdinando , Cervoni Maria Luisa , Barbarino Marcella , Guglielmi Mario Berardino , Pisano Claudio , Cabri Walter
Published: August 2012

Hsp90 (heat shock protein 90) is a component of a molecular chaperone complex, involved in the folding, maturation and stabilisation of key signalling proteins which control cell proliferation, survival and transformation. It works by modulating a set of cancer-associated proteins, that are often over-expressed and/or mutated in tumor cells, collectively referred as ‘‘clients’’. Inhibition of Hsp90 causes destabilization and eventual degradation of client proteins involved in cell cycle, tumor growth, angiogenesis and apoptosis. Therefore, their modulation results in suppression of tumor growth by multiple parallel mechanisms. Consequently, inhibition of Hsp90 is believed to be a potential target for cancer therapy, also supported by a number of molecules currently under investigation in different phase of clinical trials. We recently synthesized 1,4,5-trisubstituted-1,2,3-triazole derivatives and surprisingly found that these new molecules demonstrated to be extremely active both on the biological target and on human tumor cell lines, even at nanomolar concentration. 3D QSAR analysis was also performed in order to rationalize HSP90 binding data. The overall profile of this new class of 1,2,3-triazoles, including stereoselective synthesis and a comprehensive pharmacological in vitro characterization, will be presented.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3896. doi:1538-7445.AM2012-3896
Abstract 3897: Design and preclinical evaluation of single agents with antitubulin and antiangiogenic activity as antitumor agents
Authors: Gangjee Aleem , Pavana Roheeth Kumar , Zhang Xin , Ihnat Michael , Hamel Ernest
Published: August 2012

Agents that interfere with microtubules are important antitumor agents. Tumor angiogenic mechanisms that are vital for tumor growth and metastasis are targeted by antiangiogenic agents. Antiangiogenic agents are usually not tumoricidal but are mainly cytostatic. Combination chemotherapy with antiangiogenic and cytotoxic agents has shown significant promise, and several studies with such combinations are in progress in the clinic. Single agents with both antiangiogenic activities and cytotoxicity could afford single agents that would circumvent the pharmacokinetic problems of multiple agents, avoid drug-drug interactions, be used at lower doses to alleviate toxicity, be devoid of overlapping toxicities, and delay or prevent tumor cell resistance. We have designed, synthesized and evaluated two novel compounds, AG119 and AG425, that inhibit both vascular endothelial growth factor receptor-2 (VEGFR-2) for antiangiogenic effects and tubulin for cytotoxic effects in single agents. Both compounds have comparable potency as sunitinib and semaxinib against cell lines overexpressing VEGFR-2 and were equipotent with semaxinib on VEGFR-2 mediated endothelial cell proliferation, indicating a functional effect of these agents on VEGFR-2 inhibition (IC50: semaxanib 68.2 ± 4.2 µM, AG119: 60.2 ± 2.4 µM, AG425: 52.3 ± 3.8 µM). The novel compounds are effective and potent inhibitors (comparable to combretastatin A-4) of bovine brain tubulin assembly (IC50: combretastatin A-4 1.2 ± 0.01 µM, AG119: 3.5 µM, AG425: 1.9 ± 0.1 µM). The compounds were potent inhibitors of [3H]colchicine binding to tubulin, indicating that these compounds, particularly AG425, bind at the colchicine site. These agents cause microtubule depolymerization, arrest cells in late G2/M phase and trigger apoptotic cell death. Moreover, AG119 and AG425 are potent anticancer compounds in aggressive basal like breast cancer (BLBC) and triple negative breast cancer (TNBC) cell lines. The novel compounds showed sensitivity in taxol resistant cells, indicating that they are unlikely to be Pgp or MRP substrates [unlike taxol], and these compounds possess relative selectivity of kill on tumor versus normal cell types. In vivo these agents reduce tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 model and U251 glioma model] in immunodeficient mice, superior to docetaxel and sunitinib, without overt toxicity to the animals.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3897. doi:1538-7445.AM2012-3897
Abstract 3898: NOSH-aspirin: A novel nitric oxide- and hydrogen sulfide-releasing hybrid for treatment of colon cancer
Authors: Lee Diana H. , Nesbitt Diandra E. , Chattopadhyay Mitali , Kodela Ravinder , Kashfi Khosrow
Published: August 2012

Introduction: In our search for a better non-steroidal anti-inflammatory drug (NSAID) we have synthesized a series of nitric oxide-, and hydrogen sulfide-releasing hybrid compounds that have IC50s for cell growth inhibition in the low nano-molar range. We have termed these NOSH compounds. Our lead agent, NOSH-aspirin is in the order of 100,000 to 250,000-fold more potent than aspirin in inhibiting colon cancer cell growth over a 24-72h time period. NOSH-aspirin although highly potent, has very limited cyto-toxicity, lactate dehydrogenase (LDH) release at 4 times its IC50 for cell growth inhibition was less than 10% at 24h. Here we report our results on the effects of NOSH-aspirin treatment on established tumors in a xenograft model of human colon cancer cells. Methods: Compound: NOSH-aspirin was synthesized and purified by us with 1H-NMR verification. Cell line: HT-29 human adenocarcimoma; Xenografts: Male athymic SKID mice were implanted s.c. in the right flank with HT-29 (2 x 106) cells suspended in 50% Matrigel, after 10 days the animals were randomly divided into 4 groups (N = 7/group) and gavaged daily with NOSH-aspirin (25 or 50 or 100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 3 days. After 3 weeks of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for immunohistochemical studies. Results: NOSH-aspirin had no effect on the weight of the mice even at the highest concentration used. There were no overt signs of toxicity. It dose-dependently reduced tumor mass by 50 ± 7%, 75 ± 5%, 90 ± 3 % at 25, 50, and 100 mg/kg, respectively. Tumor volume was also dose-dependently reduced as a function of time. NOSH-aspirin inhibited growth of HT-29 colon cancer cells xenografts as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells). NF-κB, activated in control xenografts was significantly inhibited by NOSH-aspirin. Conclusions: NOSH-aspirin has potent anti-cancer affects and merits further evaluation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3898. doi:1538-7445.AM2012-3898
Abstract 3899: NOSH compounds: Nitric oxide- and hydrogen sulfide-releasing hybrids, a new class of anti-inflammatory pharmaceuticals
Authors: Vannini Federica , Kim Han-Bee , Chattopadhyay Mitali , Kodela Ravinder , Kashfi Khosrow
Published: August 2012

Introduction: The clinical usefulness of non-steroidal anti-inflammatory drugs (NSAIDs) combined with their potentially life-threatening toxicity has prompted intensive efforts to identify safer alternatives, which will at least maintain their pharmacological properties. The search for a “better NSAID” has resulted in at least two modified hybrids, one that releases nitric oxide (NO-NSAIDs), and another that releases hydrogen sulfide (HS-NSAIDs). These agents although much safer and more potent than their traditional counterparts, have IC50s for cell growth inhibition that are in the micro-molar range. Therefore, we postulated that a new hybrid that incorporated the active parts of each compound may be more potent and effective than either one. Our hypothesis has proved to be correct. We have synthesized a number of NSAID based NOSH (nitric oxide-, hydrogen sulfide-releasing) compounds that release both H2S and NO, and have IC50s for cell growth inhibition in the low nano-molar range. Methods: Compounds: Aspirin based NOSH-1, NOSH-2, NOSH-3, and NOSH-4 were synthesized and purified by us with 1H-NMR verification. Human cancer cell lines: colon (HT-29, HCT 15, SW480), breast (MCF-7, MDA-MB-231, SKBR3), pancreas (BxPC-3, MIA PaCa-2), lung (A549), prostate (LNCap), leukemia (Jurkat). Cell growth: MTT. Anti-inflammatory: paw edema induced by intraplantar injection of 100 µL of 1% carrageenan, paw volumes measured up to the tibiotarsal joint immediately prior to carrageenan injection and thereafter at 1hr intervals up to 6hrs. Drugs were administered orally 1 hr before carrageenan; COX enzyme: immunoblotting, PGE2: ELISA Results: All NOSH compounds were more effective than aspirin in inhibiting the growth of colon, breast, pancreas, prostate, lung, and leukemia cancer cell lines. The IC50s at 24h were in the range 48 nM to 6.5 µM depending on the cell line and the compound. The corresponding IC50 for aspirin was >5000 µM in all cell lines. Our lead compound NOSH-1, had a enhanced potency of >100,000-fold. NOSH-1 dose-dependently decreased the carrageenan-induced edema 1h after administration and maintained this anti-inflammatory effect throughout the experiment (up to 6h). PGE2 levels within the inflamed rat paw were also reduced by NOSH-1. Carrageenan induced both COX-1 and COX-2 expression which were dose-dependently inhibited by NOSH-1. Conclusions: NOSH compounds are a new class of anti-inflammatory agents. NOSH-1, our lead compound is very potent in inhibiting a number of different cancer cell lines of different tissue origin suggesting a tissue independent effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3899. doi:1538-7445.AM2012-3899
Abstract 39: Mir-21 regulates TGF-β1-induced CAF formation through Smad7
Authors: Mi Jun
Published: August 2012

The TGF-β1, one of major cytokines secrected by tumor cells, not only promotes the growth of tumor cells themselves, but also transforms the adjacent stroma cells into cancer-associated fibroblast formation. However, the detail mechanism by which TGF-β1 induces CAF (cancer-associated fibroblast) formation is unveiled. In this study, the primary fibroblast from skin was successfully transformed into CAF in vitro. These TGF-β1-induced CAF cells significantly promoted the tumor growth in the A175 xenografted mice model. Moreover, the TGF-β1 stimulation not only increased the amount of miR-21, but also reduced the protein level of Smad 7 in primary fibroblast. The TGF-β1 accelerated the Drosha-mediated miR-21 maturation and the Smad 7 was identified to be a target of miR-21. Both the overexpression of miR-21 and knocking down of Smad 7 induced the CAF formation, which represented the TGF- β1 triggered phenotype in primary fibroblast. The Smad 7 physically associates with the Smad 2/3 and regulates the activation of Smad 2/3. The activation of Smad signaling globally changed the status of chromatin and triggered the reprogramming process and transformed the primary fibroblast into CAF.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 39. doi:1538-7445.AM2012-39
Abstract 3902: Development of Aurora A inhibitors with ortho-halophenyl substituted pyrimidines: Unusual potency, SAR and X-ray crystallography studies.
Authors: Lawrence Harshani R. , Martin Mathew P. , Luo Yunting , Pireddu Roberta , Gevariya Harsukh , Ozcan Sevil , Zhu Jin-Yi , Rodriguez Mercedes , Elias Roy , Yang Hua , Sebti Said M. , Schonbrunn Ernst , Lawrence Nicholas J.
Published: August 2012

The Aurora kinases are a family of serine-threonine kinases that play an important role in the regulation of cell division. The three members of the Aurora family Aurora A, B and C share a high degree of structural homology in their kinase domain but each kinase plays a different role in the control of mitosis. Aurora A and B have received the most attention to date as anticancer targets. Detailed studies have shown Aurora A and B are over expressed in a variety of cancers and are implicated in many aspects of tumor development. Aurora A is frequently over-expressed in tumors and cancer cell lines and has characteristics of an oncogene. Aurora B is also over expressed in many cancer types but does not have oncogenic properties. Aurora B plays important roles in M phase to ensure correct chromosome-microtubule alignment and attachment and chromosomal cytokinesis. Therefore, inhibition of Aurora kinase A and B is emerging as target-based therapy in cancer treatment. From our previous work HLM-8598 was identified from an in-house chemical library as a potent and highly selective inhibitor for Aurora A (IC50 value of 0.073 ± 0.002 μM) over Aurora B (IC50 = 5.4 ± 1.8 μM). X-ray crystallography studies of HLM-8598 bound to Aurora A confirmed HLM-8598 is a type-1 kinase inhibitor that interacts with the ATP binding site of the enzyme. The HLM-8598 bound to Aurora A structure-assisted synthesis of new analogs with IC50 < 2.5 nM affinity. We will present the synthesis of new analogs that contain an ortho-halogen substituted phenyl moiety, structure activity relationship studies as a part of our efforts to identify unusually potent Aurora A kinase inhibitors. We will discuss the use of kinase structural elements to rationally guide compound synthesis and improve design strategy. The in vitro and in vivo activities of ortho-halogen phenyl substituted pyrimidines will be presented as unusually potent Aurora A inhibitors and potential anti-cancer agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3902. doi:1538-7445.AM2012-3902
Abstract 3905: Synthesis and structure activity relationship of substituted N,6-diphenyl-5,6-dihydrobenzo[h]quinazolin-2-amine as inhibitors of fibroblast growth factor receptors (FGFR)
Authors: Ali Syed M. , Brassard Chris , Dalton Audra , Kizer Darin , Lapierre Jean-Marc , Liu Yanbin , Moussa Magdi , Palma Rocio , Tandon Manish , Vensel David , Volckova Erika , Wang Jianqiang , Westlund Neil , Wu Hui , Yang Rui-Yang , Bates Craig , Bhavsar Mayank , Bull Cathy , Eathiraj Sudharshan , Nicewonger Robert , Savage Ron , Waghorne Carol , Castro Jennifer , Nakuci Enkeleda , Chen Chang-Rung , France Dennis S. , Chan Thomas C.K. , Ashwell Mark A.
Published: August 2012

Utilization of hydrophobic motifs present in auto-inhibited protein kinases has resulted in the identification of a series of 5,6-dihydrobenzo [h]quinazolin-2-amines with activity as fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors. Herein we describe the combination of a proprietary in silico design process, a new screening paradigm using an array of biochemical and biophysical technologies in conjunction with an established parallel chemistry process for the identification and optimization of a series of novel FGFR inhibitors. These potent FGFR inhibitors exhibit a preference for the inactive form of the kinase, are non-ATP competitive, and exhibit robust cellular pharmacodynamic inhibition as well as in vitro anti-proliferative effects in cells dependent on FGFR and significant anti-tumor activity in appropriate xenograft models in vivo. The design strategy, synthesis, structure activity relationships and in vitro and in vivo biology of selected inhibitors will be presented.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3905. doi:1538-7445.AM2012-3905
Abstract 3908: Norton-Simon modeling for the optimization of dose and schedule of palifosfamide in breast cancer
Authors: Traina Tiffany A. , Hudis Clifford , Lewis Jonathan , Norton Larry
Published: August 2012

Palifosfamide (P), the stabilized active metabolite of ifosfamide, is a bifunctional DNA alkylating agent with broad anticancer activity, including against cells with ALDH over-expression resistant to cyclophosphamide. In contrast to other agents of its class, P's favorable safety profile, rapid onset of action, and comparable activity by IV and PO routes permits optimization of dose-schedule. To this end we used Norton-Simon Modeling to analyze the growth curves of the MX-1 breast cancer xenograft model, unperturbed and perturbed by various dose-schedules of P. This method fits an equation ΔN(t)/αt=F(N)·G(t) to experimental data on tumor size N as a function of time t to define the magnitude and shape of the curve of growth perturbation G as a function of time t before, during, and after the administration of anticancer therapy. In general (as in this case), growth perturbation “peaks” at a number of days after administration and declines thereafter. This is largely due to the induction of spontaneously reversible resistance (tachyphylaxis) as evidenced by the cancers’ reversion to drug sensitivity after a period of drug absence. Hence, repeated cycles of intermittent therapy allows for further growth perturbation, providing for sustained anticancer effect. The solution of the Norton-Massague equation ΔN(t)/αt=aN^b-cN^d fit unperturbed growth exactly (r^2=0.99973). 180 mg/kg as a single dose caused tumor volume regression with a peak growth perturbation at 7 days after administration. The effect of lower dose levels in (growth perturbation as a % of that of 180 mg/kg as a single dose; peak at days after start of administration) were 120 mg/kg (75%; 7), 80 mg/kg (34%; 5), and 53 mg/kg (16%; 3). When P was given in divided doses over 5 days, 180 mg/kg cumulative dose resulted in (94%; 10), 120 mg/kg (55%; 6), and 80 mg/kg (16%; 3). Hence, the administration of P over 5 days preserved almost full efficacy but delayed the time of emergence of drug resistance by 43% (from 7 days to 10 days). Moreover, analysis of the effects of P alone, docetaxel alone, and the simultaneous combination indicates that the shapes and magnitudes of the single-agent curves add to perfectly predict that of the combination, with minimal loss of single-agent activity. Hence, the daily x 5 schedule (9 days off) at highest tolerated dose level is proposed for clinical study, both as a single agent and in combination with other anticancer medications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3908. doi:1538-7445.AM2012-3908
Abstract 3911: Infrared vibrational frequency as a novel molecular descriptor for lead optimization of PKB/Akt inhibitors
Authors: Kawakami Joel K. , Harrison Kristin , Baldauf Ashley , May Heidi
Published: August 2012

We report the use of a novel molecular descriptor used for lead optimization of PKB inhibitors. Lead optimization remains a major rate limiting step in drug discovery. Current methods for lead optimization that leads to a clinical candidate often fail during clinical trials with attrition rate as high as 9 out of 10. We re-investigated the lock and key theory by incorporating the vibrational frequency of small molecules. The vibrational frequency is calculated based on the summation of all theoretical infrared vibrational frequency of each bond and normalized via division by its molecular weight. We term this molecular descriptor as MDIR. The MDIR is utilized as a rapid lead optimization computational chemistry method. The MDIR correlation to affinity/inhibition at several molecular targets shows a parabolic relationship. Quinoxalines with MDIR values near 135 provide the best inhibition (19% INH at 10 uM) at PKB/Akt as assessed via the Cisbio HTRF KinEASE assay. An internal control, 10-DEBC, known to have 100 % inhibition of PKB at 2.5 uM, had only a 6 % INH @ 10 uM in the KinEASE assay.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3911. doi:1538-7445.AM2012-3911
Abstract 3916: Inhibition of GREB1 mRNA and protein limits ER+ breast tumor cell proliferation
Authors: Hnatyszyn H. James , Olson Rebecca , Pierce Rachel , Herbert Lynn , Lippman Marc E.
Published: August 2012

Discovering the mechanism by which estrogen regulates breast cancer growth will aid in the identification of patients who will benefit from endocrine therapy. Estrogen (E2) binding to the estrogen receptor (ER) stimulates the proliferation of normal mammary cells and some breast cancers. Preventing ER expression or receptor-ligand interaction in hormone-responsive breast cancers inhibits cell division and promotes tumor cell death. However, the mechanism of E2-regulated breast cancer cell growth remains unclear. Although E2-regulated genes have been identified, those critically involved in growth regulation remain elusive. Identification of critical E2-regulated genes involved in mammary cell proliferation would elucidate the key pathway(s) supporting hormone-mediated tumor growth as well as provide insight into mechanisms of resistance to endocrine therapies and potential prognostic and therapeutic targets for ER+ breast cancer treatment. Gene regulated in breast cancer 1 (GREB1) is an estrogen-regulated gene that has been implicated in hormone-stimulated cell proliferation and is a candidate clinical marker for response to endocrine therapy. GREB1 mRNA and protein expression correlate with ER expression in breast tumors and the addition of estrogen to ER+ breast cancer cell lines leads to an increase in GREB1 expression and cell proliferation. The function of GREB1 and its role in ER+ breast tumors remains undefined. Experiments were designed to elucidate the role of GREB1 in ER+ breast cancer cell lines. ER+ breast cancer cell lines were transfected with GREB1-specific siRNA and control species. These tumor cell populations were evaluated for GREB1 mRNA and protein expression, cell growth and proliferation as well as invasion and migration in vitro. Sequence-specific siRNA effectively inhibited GREB1 mRNA and protein expression for more than 96 hours in MCF7 and T47D ER+ breast tumor cell lines. Utilizing the real-time Xcelligence system to monitor cell growth in culture suggested tumor cells treated with siRNA targeting GREB1 had significantly retarded cell proliferation. These observations suggest that GREB1 is a critical E2-regulated gene involved in breast tumor cell proliferation and is an exploitable target for novel breast cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3916. doi:1538-7445.AM2012-3916
Abstract 392: The role of post surgical inflammation in tumor progression in breast cancer
Authors: Rincon Mercedes , James Ted , Bunn Janice
Published: August 2012

Introduction The process of chronic inflammation inducing malignant transformation is well known and recognized in several disease states, including ulcerative colitis (colorectal carcinoma), chronic wounds (squamous cell carcinoma), and hepatitis B infection (hepatocellular carcinoma) as common examples. Recent evidence suggests that this process may be relevant in other tumor types and may be influenced by inflammation arising secondary to interventional procedures. The purpose of this study is to investigate the potential role of inflammatory mediators resulting from the post-surgical healing process on tumor proliferation and progression to metastasis in breast cancer. Methods We used MMTV-PyV MT female mice (transgenic mice expressing the middle T antigen of the polyomavirus under the control of the MMTV promoter/enhancer) as a mouse model of human epidermal breast cancer. Mice underwent a surgical punch biopsy (2 mm) in the center of the tumor, or underwent the same procedure (transient anesthesia) without biopsy. Mice were paired according to the tumor size at the time of biopsy. Mice were then euthanized 8 days after biopsy, and lungs were harvested and histologically examined for the presence of micrometastases. To further evaluate the effect of inflammation in the development of metastases, a separate subset of mice were treated with ibuprofen versus vehicle (placebo control) for three days after biopsy. Results No substantial difference in the primary tumor growth was observed after biopsy between the various groups. However, a greater number of metastases were found in the lungs of mice that underwent biopsy relative to the number in mice that did not receive biopsy. The number of metastases was reduced in the set of mice receiving ibuprofen treatment following the biopsy procedure (9.3 vs. 25 p<0.05). Conclusion Our study provides evidence to suggest that inflammatory mediators arising secondary to the healing process may stimulate tumor proliferation and metastasis. These findings may hold significance for the timing of breast cancer treatment following diagnostic needle biopsy, as well as for the potential impact of excisional biopsy on the outcome of patients with breast cancer. Further research into the mechanisms of this interaction may help to increase our understanding of breast cancer biology and may yield new strategies (e.g. anti-inflammatory medication) to optimize treatment and outcome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 392. doi:1538-7445.AM2012-392
Abstract 3920: Off-target effects of the putative G-protein coupled estrogen receptor 1 (GPER) agonist G1 in ovarian and breast cancer cells
Authors: Wang Cheng , Jiang Chao , Lv Xiangmin , Fu Lan , Davis John S.
Published: August 2012

G-protein coupled estrogen receptor 1 (GPER or GPR30) plays important roles in mediating estrogen action in many different tissues and organs under both physiological and pathological conditions. G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone), the putative selective GPER agonist, has been reported to activate GPER, trigger several signaling pathways, and stimulate cell proliferation in ER-negative breast cancer, endometrial and ovarian cancer. More recently, reports showed that activation of GPER by estrogen or G1 can suppress certain cell proliferation. This study was initiated to clarify the function of GPER and its agonist on the granulosa cell tumor (GCT) cell proliferation using KGN cell as a cellular model. Well characterized epithelial ovarian cancer and breast cancer cell lines were used for comparison. GPER siRNA was used to knock down GPER. Western blot and immunohistochemistry were used to detect protein expression. We found that knockdown of GPER significantly (P < 0.05) suppressed KGN cell proliferation, suggesting that GPER may play positive roles on KGN cell proliferation. Low concentrations of G1 (<500nM) had no effect on KGN cell proliferation, whereas higher concentrations (>1μM) of G1 significantly suppressed KGN cell proliferation and arrested KGN cells in G2/M phase. These results indicated that G1 may suppress KGN cell proliferation in a GPER-independent manner. Supporting our hypothesis, knockdown of GPER did not block the inhibitory effect of G1on KGN cell proliferation. Moreover, G15, a potent GPER antagonist, failed to block G1-induced suppression of cell proliferation and G1-induced cell cycle arrest in KGN cells. In IGROV-1 epithelial ovarian cancer cells, G1 suppressed cell proliferation, a response which was not blocked by G15, even at concentrations 8-fold higher than that of G1. G1 treatment also suppressed the proliferation of MDA-MB-231 breast cancer cells, a cell line which reportedly expresses very low amounts of GPER. In HEK-293 cells, which are reported to have no expression of GPER, G1 also significantly suppressed the cell proliferation and induced cell cycle arrest. In conclusion, our results demonstrate that G1 is able to suppress ovarian and breast cancer cell proliferation in a GPER-independent manner. G1 may be a candidate for the development of drugs against ovarian and breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3920. doi:1538-7445.AM2012-3920
Abstract 3923: Growth hormone activates ERK via EGFR, not IGF-IR, to potentiate estrogen-dependent breast cancer cell proliferation
Authors: Felice Dana L. , Unterman Terry G. , Swanson Steven M. , Frasor Jonna
Published: August 2012

Estrogen receptor (ER) and insulin-like growth factor-I receptor (IGF-IR) interact and activate one another in breast cancer cells, and ER can up-regulate components of the IGF-I signaling pathway. Therapeutic strategies co-targeting ER and IGF-IR in ER-positive breast tumors have been developed, yet clinical trials of anti-IGF-IR therapies have not been as successful as had been hoped. Although growth hormone (GH) is implicated in having a role in breast cancer, its effects are often attributed to the actions of IGF-I. Since inhibition of IGF-IR can cause an increase in circulating levels of GH, we considered the possibility that GH may act directly on the tumor itself thus explaining the limited efficacy of anti-IGF-IR therapy in breast cancer. Therefore, we sought to determine whether GH has direct, IGF-I-independent effects on breast cancer. We demonstrate in T47D human breast cancer cells that while GH alone only weakly stimulates cell proliferation, it significantly enhances estradiol (E2)-stimulated proliferation. Inhibition of IGF-IR reduced E2-stimulated proliferation, confirming previous findings. Remarkably, GH+E2 overcame the IGF-IR blockade to promote proliferation. This indicates that GH not only acts in an IGF-I-independent manner, but can bypass IGF-IR inhibitors to restore E2 action on proliferation. Furthermore, we found that both epidermal growth factor receptor (EGFR) and ERK were required for proliferation stimulated by GH and E2, and that GH uses EGFR to activate ERK even in the presence of the IGF-IR inhibitor. Expression of proliferation genes up-regulated by GH+E2 treatment was abrogated by the EGFR and MEK inhibitors, but not by the IGF-IR inhibitor. Thus, we propose that while E2-stimulated proliferation requires IGF-IR, EGFR, and ERK, the presence of GH allows E2 to bypass the requirement for IGF-IR while remaining dependent on EGFR and ERK. Furthermore, we have found that GH can potentiate E2 stimulation of ER target gene transcription and this is dependent on ERK, but not on IGF-IR. Taken together, these findings indicate that GH not only can act directly on breast cancer cells independently of IGF-I, but can bypass IGF-IR blockade to stimulate ERK and potentiate E2 activity on proliferation and gene regulation. A better understanding of the interactions between GH and E2 will contribute to the development of novel drugs or improved combination therapies targeting the GH/IGF/E2 axis in breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3923. doi:1538-7445.AM2012-3923