bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 475: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton and migration of glioma initiating cells
Authors: Alapati Kiranmai , Dasari Venkata Ramesh , Gopinath Sreelatha , Malla Rama Rao , Gujrati Meena , Rao Jasti S.
Published: August 2012

Glioblastoma (GBM) remains one of the most fatal and least successfully treated solid tumors. It is evident that some cancer cells with stem cell properties (known as glioma initiating cells; GICs) survive conventional treatments and are involved in the recurrence of tumors. In the present study, CD133+ GICs were isolated from established U251 and 5310 glioma cell lines and subjected to radiation and transfection with shRNA against uPAR and cathepsin B so that we could study the events related to cell migration. Simultaneous downregulation of uPAR and cathepsin B (pUC) caused the disruption of radiation-induced complex formation of pPKC∈/α:integrinβ1 and PKC∈:integrinβ1 in glioma cells as well as in GICs as was observed by immunoprecipitation analysis. Further, pUC treatment also inhibited the PKC/integrin signal through FAK by disintegrating the association of FAK and the cytoskeletal molecules vinculin and α-actinin. It was also observed that inhibition of radiation-induced ERK phosphorylation by pUC directed a negative feedback mechanism over the FAK signaling molecules that led to an extensive reduction in the cytoskeletal organization, and thereby generating a migratory arrest. Further confirmation was obtained by treating the cells with U0126 (10 µM), a MEK inhibitor, and rottlerin (200 µM), a PKC inhibitor. pUC treatment efficiently inhibited the co-localization of pPKC∈/α/integrinβ1, PKC∈/integrinβ1, FAK/vinculin and FAK/α-actinin in the brain tumor sections of mice implanted with glioma cells and GICs. In conclusion, shRNA-mediated knockdown of uPAR and cathepsin B could be an effective treatment strategy for controlling highly invasive glioma cells and treatment-resistant glioma initiating cells.

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 475. doi:1538-7445.AM2012-475
Abstract 4751: Structure and inhibition of mitogen-activated protein kinase kinase 4 (MEK4): A prostate cancer pro-invasion protein
Authors: Krishna Sankar Narayan , Luan Chi-Hao , Minasov George , Shuvalova Ludmilla , Farmer Rebecca L. , Nibbs Antoinette , Scheidt Karl A. , Anderson Wayne F. , Bergan Raymond C.
Published: August 2012

Introduction: Metastasis of Prostate Cancer (PCa) represents the second highest cause of death due to cancer among men in the United States. If this unregulated movement of cells can be inhibited, mortality arising from PCa will be greatly reduced. Our group has shown that Mitogen-activated protein kinase kinase 4 (MAP2K4/MEK4), a 399 amino acid protein, activates pro-invasion signaling pathways in human PCa. Further, we have shown that 4,5,7-trihydroxyisoavone (genistein) inhibits MEK4 kinase activity, cell invasion and metastasis of human PCa cells in a murine model. While therapeutically effective, genistein represents a non-chemically optimized natural product with additional undesired effects such as estrogenic receptor stimulation. Recognizing that MEK4 represents a novel and important therapeutic target, we have sought to characterize its structure, biochemical function and have synthesized genistein analogs designed to target it. Summary of Results: Using X-ray crystallography we have obtained a 3.38Å resolution structure of the MEK4 catalytic domain. We are in the process of improving our resolution, and of deriving 3D structures with inhibitors bound to MEK4. We have evaluated biophysical interactions between MEK4 and ligands (i.e., genistein and its analogs). Ligand binding to a protein tends to increase its stability, thereby increasing the temperature of denaturation, or melting. Using a fluorescence thermal shift (FTS) assay, we demonstrated that genistein and 6 of 39 novel analogs thermally stabilized MEK4 in a concentration dependent manner, consistent with MEK4 binding. We identified a similar pattern in 5 compounds from a commercial library, one of which was staurosporine - a broad spectrum protein kinase inhibitor. We developed and characterized a novel in vitro MEK4 kinase assay, using kinase dead p38MAPK (K53A) as substrate. In human PCa cells, MEK4 activates the p38 MAPK pro-invasion pathway. To date, we have used this assay system to demonstrate that staurosporine inhibits MEK4 with nanomolar IC50 values. Conclusions and Future Directions: MEK4 is a novel target for therapeutic intervention in PCa. Our ultimate goal is to synergistically utilize structural and biochemical information to inform the synthesis of inhibitors that specifically and selectively act upon MEK4 to inhibit human PCa metastasis in man.

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 4751. doi:1538-7445.AM2012-4751
Abstract 4752: Selective inhibition of cancer metastasis with a novel small therapeutic molecule
Authors: Xu Li , Farmer Rebecca , Huang Xiaoke , Pruell Sean , Voll Eric , Pavese Janet , Valsecchi Matias , Biddle Margaret , Nibbs Antoinette , Avram Michael , Scheidt Karl A. , Bergan Raymond C.
Published: August 2012

Prostate cancer (PCa) is the second most common cause of cancer death in US males. Death is typically caused by metastasis. Naturally occurring isoflavanones have been reported to be a class of compounds that effectively inhibit PCa motility and metastasis. This led us to use these compounds as a synthetic scaffold starting point. By integrating fragment-based diversification synthesis with chemi-driven biological selection, we discovered novel small molecule therapeutics with increased selectivity and potent efficacy. We thereby efficiently synthesized a new class of bioactive compounds that inhibit cell motility in vitro and inhibit human PCa metastasis in a murine model at low nanomolar concentrations. Extensive investigations indicate high specificity at the molecular and cellular levels, and failed to identify toxicity, even at high doses administered over extended periods. Importantly, efficacy against several cancer types was also demonstrated. Target validation studies used our lead as a chemical probe, and point to inhibition of ATM/ATR interaction with specific substrate proteins as important. Together, these studies indicate that we have successfully discovered a novel compound, acting upon a novel pharmacologic target, which selectively inhibits human PCa metastasis. Taken with our favorable preclinical toxicological data, these findings support movement of our lead compound into early phase human trials.

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 4752. doi:1538-7445.AM2012-4752
Abstract 4753: Targeting nucleotide excision repair: Chemical synthetic lethality and biology-based combination therapy
Authors: Turchi John J. , Lee Jiyoon , Hendricks Jeremiah , Neher Tracy M. , Fitch Richard W.
Published: August 2012

As DNA repair defects in cancer can be exploited for therapeutic benefit via synthetic lethality, new DNA repair targets and compounds to inhibit them will expand the armamentarium against cancer. While most targeted therapies involve disrupting enzyme-substrate interactions we have pursued targeting protein-DNA interactions to validate an entire new class of molecular interactions that can be targeted with small molecule drugs. We have focused on the nucleotide excision repair (NER) pathway and two proteins, the single-stranded DNA binding protein, replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein. We have identified and developed small molecule inhibitors (SMIs) of each target. In vitro analysis of XPA SMI has led to a 50-fold increase in potency and revealed structure-activity relationships that define the important pharmacophores for XPA interaction. Both reversible and irreversible SMIs of RPA have been identified and cellular analysis of irreversible RPA SMI's revealed single agent anticancer activity at sub-micro-molar concentrations. The degree of the anticancer effect of cellular RPA inhibition varied among cancer types and was dependent on the DNA repair phenotype. Synergy between NER inhibition and cisplatin will be presented and as a function of DNA repair phenotype. These data demonstrate the ability to develop SMI of protein-DNA interactions that impact DNA repair and can be employed to increase the sensitivity of repair deficient tumors to DNA damaging therapeutics. This works was supported by NIH grant R01-CA82741 to JJT

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 4753. doi:1538-7445.AM2012-4753
Abstract 4760: Inhibition of c-myc by the triterpenoid celastrol
Authors: Hu Angela , Wang Huabo , Pendleton Kelsey , Prochownik Edward V.
Published: August 2012

c-Myc is a bHLH-ZIP transcription factor that is deregulated in a variety of human cancers. More specifically, c-Myc heterodimerizes with another bHLH-ZIP partner protein, Max, to bind to specific sequences (E-Boxes: CAC/TGTG) located near the transcriptional start sites of its numerous target genes. Because c-Myc's highly variable downstream effects are difficult to attack individually, directly inhibiting the association between c-Myc and Max association by interfering with their bHLH-ZIP-mediated heterodimerization has emerged as an attractive therapeutic strategy. Thus far, high throughput screens of chemical libraries have identified a number of small molecules that specifically inhibit the c-Myc-Max association. However, these synthetic compounds and their analogs are generally of low potency and therefore have limited clinical utility. Here we report identification of celastrol, a naturally occurring triterpenoid, as a potent c-Myc inhibitor. We sought to further assess celastrol's ability to inhibit c-Myc-Max interaction and cell growth, and determine its structure activity relationship. Surface plasmon resonance and electrophoretic mobility shift studies revealed that celastrol specifically binds to c-Myc and prevents both its interaction with Max, and subsequent DNA binding with a Kd in the low micromolar range. Co-immunoprecipitation studies confirmed that the dose-dependent disruption of c-Myc-Max heterodimers by celastrol could also be achieved in c-Myc-overexpressing human HL60 promyelocytic leukemia cells. Moreover, in vitro data demonstrated that celastrol significantly inhibited both HL60 and Burkitt's lymphoma cell growth with IC50s in the nanomolar range. However, modifications to the C-28 carboxylic acid group of celastrol eliminated c-Myc binding, suggesting that celastrol binds to c-Myc via this functional group. This structure activity relationship may provide a basis for the development of more pharmacologically suitable analogs. Together these results suggest that celastrol is among the most potent Myc-Max disruptors yet identified, and suggest a mechanism of action. Celastrol thus represents a promising new agent with which to target c-Myc and provides a new structural chemical platform for further pharmaceutical 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 4760. doi:1538-7445.AM2012-4760
Abstract 4763: Targeting p300 for inhibiting triple negative breast cancer
Authors: Liao Daiqing , Yang Heng , Luo Jian
Published: August 2012

Breast cancer (BC) can be classified into four main subtypes: normal-like, HER2 overexpressing, luminal A and B, and basal-like BC. Normal-like BC cells exhibit gene expression pattern resembling basal epithelial cells. Erb-B-2/HER2-positive BC cells display high levels of Her2 expression. Luminal tumors show heightened expression of luminal cytokeratins and genes specific to the luminal epithelial cells, and are generally ER positive. Basal-like BC is characterized by the lack of expression of ER, progesterone receptor (PgR), and HER2. Triple-negative BC (TNBC) is defined by the lack of expression of ER, PgR and HER2 with distinct molecular, histological and clinical characteristics. Among the four BC subtypes, TNBC shares many of the molecular features of basal-like BC. Patients with TNBC have worse prognosis than those with other types of BC due to residual diseases that are resistant to chemotherapy and display heightened tendency to disseminate to distant organs. Available targeted therapies are ineffective for TNBC. Interestingly, cells derived from TNBC contain an elevated proportion of tumor cells with stem cell traits. These cells, known as cancer stem cells (CSCs), can self renew, differentiate into other types of cells in the tumor bulks, and efficiently promote tumor regrowth. CSCs are highly resistant to chemotherapy due to the expression of antiapoptotic genes. Several highly active signaling pathways, including cytokine-JAK-STAT3, Wnt-β-catenin, and Hippo-TAZ cascades, support the survival and sustained self-renewal of CSCs. One remarkable feature of these diverse signaling pathways is the reliance on the functions of the coactivators and acetyltransferases p300 and its paralog CBP for the expression of the effector genes that underlie the CSC phenotypes. We therefore hypothesize that inhibition of p300/CBP is an effective strategy for treating TNBC. Using an unbiased ultra high-throughput screening, we have identified small chemical compounds that were highly toxic to TNBC cell line MDA-MB-231 and potently inhibited the enzymatic activity of p300. Validation experiments revealed that a specific compound, L002, with the IC50 of 1.98 µM against p300 in vitro suppressed histone acetylation at several known sites of p300 acetylation in cell-based assay. L002 was highly toxic to MDA-MB-231 cells but much less so to MCF7 derived from a luminal BC. Experiments are underway to examine the effects of L002 on the survival of breast CSCs. (This work was supported by Bankhead-Coley Cancer Research Program of the Florida Department of Health.)

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 4763. doi:1538-7445.AM2012-4763
Abstract 4768: Design and development of selective sphingosine kinase 2 inhibitors
Authors: Liu Kai , Zhang Shijun
Published: August 2012

Sphingosine-1-phosphate (S1P) has been recently implicated to play a vital role in regulating a variety of physiological and pathological processes including proliferation, differentiation, migration, and cell survival. S1P is produced endogenously by sphingosine kinases (SKs) and the balance of the levels of S1P, its precursor sphingosine, and ceramide, referred to as the “sphingosine rheostat,” is important for normal cellular functions. To date, two isoforms of SPKs have been reported, sphingosine kinase 1 (SK1) and sphingosine kinase 2 (SK2). Many efforts have been invested to study the roles of SK1 and SK1 has been indicated in the regulation of cancer progression and development. However, much is still remain unknown for SK2 and current results of the function of SK2 are complex and controversial. This is also reflected in the development of SK inhibitors. Several potent selective SK1 inhibitors have been reported and tested as potential cancer treatment agents. However, no potent and selective SK2 inhibitors have been developed to date. Therefore, there is an urgent need to develop SK2 selective inhibitors as pharmacological tools to help unravel the roles of SK2. Recently, we discovered that a series of thiazolidine-2,4-dione analogs selectively inhibit SK2 and one lead compound was identified to inhibit SK2 with sub-µM potency but no activity to SK1 with a concentration up to 10 µM. Herein, we will report the structure activity relationship (SAR) studies of this newly identified lead SK2 inhibitor to optimize its potency and selectivity towards SK2. Furthermore, these analogs will be biologically characterized in human leukemia U937 cells.

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 4768. doi:1538-7445.AM2012-4768
Abstract 4769: Combinatorial effect of thermally targeted p21 polypeptide and bortezomib in prostate cancer
Authors: Mikecin Ana-Matea , Syed Mariam , Raucher Drazen
Published: August 2012

Prostate cancer is the most common non-skin malignancy in men and is responsible for more deaths than any other type of cancer, except lung cancer. Most cases of prostate cancer are androgen dependent. However, during androgen ablation therapy androgen-independent prostate cancer (AIPC) cells are selected via multiple mechanisms. Since, AIPC cells are immune to androgen deprivation their appearance represents a major obstacle in the treatment of prostate cancer. Several mechanisms used by prostate cancer cells require proteasome function among which is androgen receptor recycling as well as degradation of NK-KB inhibitor IKB. Bortezomib, a FDA-approved proteosome inhibitor for the treatment of multiple myeloma was shown to induce growth arrest and apoptosis in many tumor types including AIPC, where it was shown to have a modest effect as a single treatment regime. Previously in our laboratory, Bac-ELP-p21 was found to inhibit the proliferation of SKOV3 ovarian cancer cells and induce S-phase arrest as well as apoptosis. Driven by the need for new therapeutic means for AIPC, we investigated the inhibitory effects of the p21 mimetic peptide on the growth of the androgen independent prostate cancer cell lines PC3 and DU145 as well as its potential to be used as a combination therapy with Bortezomib. For our prostate cancer research, we have further optimized the delivery system in order to get higher yields of purified protein as well as better temperature response. The redesigned p21-ELP1-Bac polypeptide retained its ability to enter both PC3 and DU145 cells and localize into the nucleus, which was confirmed using confocal microscopy and flow cytometry, respectively. Using MTS assay, we have shown that the IC50 value of both Bortezomib and the p21 mimetic peptide decreased by 1.5 and 4.5 times, respectively, when those two drugs were used in combination in both PC3 and DU145 prostate cancer cell lines. Furthermore, the combination treatment lead to an increase in phosphatidylserine exposure in both cell lines observed using flow cytometry after staining with Alexa 488 Annexin and propidium iodide. Using a BrdU incorporation assay combined with PI staining, we found that treatment with p21-ELP-Bac after treatment with Bortezomib caused an increase in the G1 phase of arrested cells in both cell lines. The thermally responsive macromolecular carrier combines the advantages of thermal targeting to the tumor site with targeting of a specific molecular pathway. Our results show that small molecule proteosomal inhibition combined with p21-ELP-Bac represents a promising AIPC treatment that can be further utilized with other therapeutic peptides.

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 4769. doi:1538-7445.AM2012-4769
Abstract 4771: Improved antitumor efficacy of an acid-sensitive doxorubicin derivative delivered by thermally responsive elastin-like polypeptides
Authors: Walker Leslie R. , Kratz Felix , Raucher Drazen
Published: August 2012

Doxorubicin is a widely used anticancer drug used in the treatment of various cancers. It acts as an intercalator of DNA and inhibits topoisomerase II thus preventing cell replication. Its side effects include nausea, myelosuppression, alopecia, and dose-limiting cardiotoxicity. Dox is nonspecific, and high doses are needed to treat cancer cells. These high doses, however, are also harmful to normal cells. Because of this toxicity to normal cells, there is a great need for a delivery system that will target dox to the tumor site. In our research, we have developed a tumor targeted drug delivery system comprising the elastin-like polypeptide (ELP) which is a thermally responsive macromolecule that remains soluble in physiologic temperature but aggregates upon heating at 42 °C in the tumor. The N-terminus of ELP was modified with the cell-penetrating peptide SynB1. The SynB1-ELP construct was further modified by the addition of an acid-sensitive hydrazone derivative of doxorubicin (DOXO), by conjugation with a 6-maleimidocaproyl moiety on the drug to a terminal cysteine residue on ELP. SynB1-ELP-DOXO was found to have a thermal transition temperature of 41 °C. This construct was 2-fold more effective in inhibiting MCF-7 breast cancer cell proliferation at 42 °C than at 37 °C, indicating that the hyperthermia-induced phase transition of ELP increases the local concentration of the drug. This inhibition may be due to apoptosis since apoptosis was enhanced 2.3-fold in MCF-7 cells when SynB1-ELP-DOXO aggregation was induced by hyperthermia. SynB1-ELP-DOXO had a similar accumulation inside the cell as free Dox as shown by confocal fluorescence imaging, and the uptake into the cell was increased by hyperthermia. In the in vivo studies, a marked reduction in tumor size was seen with SynB1-ELP-DOXO exposed to hyperthermia in the E0771 syngeneic breast cancer model in C57BL/6 mice. The efficacy of the construct was further proved by the animal's body weight increasing over time as compared to treatment with free Dox and a decreasing body weight. An ongoing study uses the combination of free Dox and SynB1-ELP-DOXO at lower doses to reduce tumor size. The ELP macromolecule can also be applied to other small molecule therapeutics opening a potential platform technology for this thermally responsive biopolymer.

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 4771. doi:1538-7445.AM2012-4771
Abstract 4772: Novel drug discovery approach targeting STAT3 for breast cancer therapy using MLSD and drug repositioning
Authors: Lin Li , Li Huameng , Xu Minghao , Zhao Zhenjiang , Olson Veronica , Xu Yufang , Li Chenglong , Lin Jiayuh
Published: August 2012

Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in human cancer, including breast cancer, and has emerged as a viable molecular target for breast cancer treatment. Novel therapeutic approaches are needed for breast cancer if significant improvements in survival are to occur. To date, few STAT3 inhibitors are available for the therapy of breast cancer. Conventional high throughput screening and virtual docking drug discovery approaches identified many hits, but only a few of them may be developed into drugs. To accelerate STAT3 drug development, we used a novel drug discovery approach with multiple ligand simultaneous docking (MLSD) which we have developed and drug repositioning to search against FDA approved drug and experimental drug database in DrugBank. Using this new method, we identified the FDA-approved drug celecoxib (a COX-2 inhibitor) with a novel function to target STAT3 [Predictive STAT3 binding dissociation constant (Kd)= 8.6 μM]. In addition, a novel celecoxib analog (8A) was developed exhibiting stronger STAT3 binding (Predicted Kd= 0.12 μM, 71-fold stronger than celecoxib). Although 8A has stronger STAT3 binding, it has weaker COX-2 binding (521-fold weaker than celecoxib), suggesting its selectivity on STAT3 versus COX-2. Minimal COX-2 inhibition by 8A may reduce cardiovascular and gastrointestinal risks associated with COX-2 inhibitors. The ability of 8A to inhibit STAT3 was tested through a combination of Western Blot, MTT cell viability assay, and Immunofluorescence Standing. 8A inhibited the P-STAT3 stimulated by IL-6 but not the induction of STAT1, STAT2, STAT4, STAT5, and STAT6 phosphorylation stimulated by IFN-γ, IFN-γ, and IL-4. 8A also inhibited the induction of STAT3 nuclear localization by IL-6, which could prevent STAT3 function as a transcription factor. 8A inhibited STAT3 phosphorylation and resulted in the induction apoptosis on breast cancer cells as evidenced by caspase-3 cleavage. Further, 8A has a lower IC50 than celecoxib and was more potent than celecoxib to inhibit STAT3 phosphorylation and induce apoptosis in breast cancer cells, which confirmed the stronger STAT3 binding energy than celecoxib. In summary, our study is the first report using MLSD and drug repositioning to develop drug-like STAT3 inhibitors. This novel method has a potential to significantly speed up the STAT3 drug development for the therapy of breast cancer and possibly other types of human cancer which express constitutive STAT3 signaling.

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 4772. doi:1538-7445.AM2012-4772
Abstract 4775: Compound S2AMN: A potential non-toxic anti-cancer therapy
Authors: Elnaggar Atif M. , Peterson Ellen C. , Kuttab Rani , Robison Richard A. , O'Neill Kim L.
Published: August 2012

S2AMN is a natural compound; garlic, onion and green pepper extracts that demonstrate apoptotic potential against a variety of cancer cell lines. In this study, we examined specifically whether treatment with S2AMN would affect the viability of several human cell lines, including Burkitt's lymphoma (Raji cell), melanoma cell lines (MDA-MB-435), human breast adenocarcinoma cell line (MCF-7) and human colon carcinoma (HT29). It was determined by flow cytometry that S2AMN works by arresting cell-cycle progression in the G1 phase. Using RT-PCR, the p53 gene was shown to be up regulated in the cells during G1 phase, suggesting that S2AMN is p53 dependent. Using DNA extraction and agarose gel electrophoresis the DNA of cells treated with S2AMN was found partially degraded after 24 hours and completely degraded after 48 hours. Viability analysis showed a dose-dependent growth curve; 24 hours after treatment there was a marked decrease in viability and within 48 hours there was complete cell death (control viability > 94%). Results from viability analyses supported the same conclusion as the DNA electrophoresis data, namely that within 48 hours there was complete DNA degradation and apoptosis. We also examined the proliferation rate at 24 and 48 hours, and found that proliferation decreased at 24 hours and completely stopped at 48 hours. One of the more striking characteristics of S2AMN is its cancer-specific cytotoxicity. After treating human lymphocytes induced with PHA (normal cells) with the same concentrations of S2AMN as shown above, cell viability, proliferation, cell cycle flow cytometry, and RT-PCR experiments were performed. Results showed that there was no significant change between treatment and control cells. In conclusion, S2AMN seems to be selectively cytotoxic by inducing apoptosis in cancer cells but not in normal cells. S2AMN could potentially be used as a non-toxic anti-cancer therapy, though more research is necessary.

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 4775. doi:1538-7445.AM2012-4775
Abstract 4778: Computer-assisted molecular design use for the discovery and optimization of rationally-designed anticancer natural products. Discovery of olive oil-derived oleocanthal as a c-Met inhibitor and optimization of Veratrum alkaloids as Hedgehog signaling inhibitors
Authors: Sayed Khalid A. El , Elnagar Ahmed Y. , Khanfar Mohammad
Published: August 2012

Dysregulation of the proto-oncogene receptor tyrosine kinase c-Met, which encodes the high-affinity receptor for hepatocyte growth factor (HGF) and the Hedgehog (Hh) pathway aberrantly associated with numerous malignancies including basal cell carcinoma, medulloblastoma, prostate, pancreatic and breast cancers. The Mediterranean diet is associated with lower incidences of cardiovascular disease, age-related cognitive disease and cancer. (-)-Oleocanthal is a naturally occurring minor secoiridoid from extra-virgin olive oil with potent anti-inflammatory activity via its ability to inhibit COX-1 and COX-2 and anti-Alzheimer's activity. Cyclopamine and its 11-oxo derivative jervine are C-nor-D-homosteroidal alkaloids with known potent teratogenic effects that induce cyclopic malformations in sheep. The jervane Veratrum alkaloids cyclopamine and jervine and the veratrane alkaloid veratramine were among the early known natural products that blocked the expression of the Hh pathway targets PTCH1 and GLI1, lowering Bcl2 levels and inducing apoptosis in many tumors. Computer-assisted molecular design (CAMD) identified oleocanthal as a potential virtual c-Met inhibitor hit. Oleocanthal inhibited the proliferation, migration, and invasion of the epithelial human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively. Oleocanthal demonstrated anti-angiogenic activity and inhibited the phosphorylation of c-Met kinase in vitro. CAMD study of our library of natural, biocatalytic, and semisynthetic jervane and veratrane alkaloids suggested that compounds with C-3-keto functionality with α4,5 or β1,2 and γ4,5 exhibited high virtual Hh binding affinity scores and possible Hh pathway activity. These compounds showed significant ability to inhibit the growth, proliferation, and migration of the prostate metastatic cancer cell line PC-3, which aided the establishment of a preliminary structure-activity relationship. (-)-Oleocanthal and Veratrum alkaloids can have potential therapeutic application for the control of c-Met- and Hh-dependent malignancies.

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 4778. doi:1538-7445.AM2012-4778
Abstract 4780: Discovery of novel checkpoint kinase 1 inhibitors by virtual screening based on multiple crystal structures
Authors: Li Yan , Kim Dong Joon , Ma Weiya , Lubet Ronald , Bode Ann M. , Dong Zigang
Published: August 2012

Checkpoint kinase 1 (Chk1) is activated in response to DNA damage through the ATM/ATR pathway, and plays an essential role in the regulation of the G2/M checkpoint. Inhibition of Chk1 should abrogate this checkpoint in a manner relevant to the treatment of cancer, and may induce apoptosis. Currently about 50 crystal structures of Chk1 are available in RCSB Protein Data Bank. The abundance of Chk1 structures offers the opportunity not only to discover new inhibitors with receptor-based drug design but also to explore the impact of receptor flexibility in ligand docking. Here we describe a successful application of the docking of multiple structures to discover novel and potent Chk1 inhibitors. Forty-six Chk1 structures were first compared in single structure docking by predicting the binding mode and recovering known ligands. Combinations of different protein structures were then compared by recovery of known ligands and an optimal ensemble of Chk1 structures were selected. The chosen structures were used in the virtual screening of over 60,000 diverse compounds for Chk1 inhibitors. Six compounds ranked at the top of the hits list were tested experimentally and two of these compounds inhibited Chk1 activity-the best with an IC50 value of 9.6 µM. Further study indicated that achieving a better enrichment and identifying more diverse compounds was more likely using multiple structures than using only a single structure even when protein structures were randomly selected.

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 4780. doi:1538-7445.AM2012-4780
Abstract 4784: Rapid mass spectrometric profiling of blood sera detects cancer with high accuracy
Authors: McDonald John F. , Fernandez Facundo , Gray Alexander , Benigno Benedict , Daneker George , Shah Nikhil , Laungani Rajesh , Tran Long , Zhou Manshui , Jones Christina , Walker DeEtte
Published: August 2012

Cancer diagnosis is problematic because the disease is often asymptomatic at early stages of progression and/or recurrence. We report here the integration of a mass spectrometric metabolic profiling procedure coupled with a support vector machine computational method for use in early cancer diagnostics, and describe successful application of the method to ovarian, pancreatic and prostate cancers.

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 4784. doi:1538-7445.AM2012-4784
Abstract 4794: Kras mutation is associated with elevated myeloblastin activity in human lung adenocarcinoma
Authors: Hillinger Sven , Wiedl Thomas , Collaud Stephane , Arni Stephan , Schraml Peter , Soltermann Alex , Moch Holger , Weder Walter
Published: August 2012

Background Lung cancer is the leading cause of all cancer deaths worldwide with suboptimal prognosis and treatment options. Therefore we aimed to identify molecular characteristics with a predictive clinical utility that at the same time might represent novel therapeutic targets for human lung adenocarcinoma. Within this study we investigated KRAS mutations, a gene frequently mutated in lung adenocarcinoma and their association with enzymatic activities of members of the serine hydrolase superfamily, a large class of enzymes that have previously been linked to cancer. Methods Participating individuals underwent surgery for lung adenocarcinoma at the University Hospital Zurich between 2003 and 2006. Formalin fixed, paraffin embedded as well as snap-frozen lung adenocarcinoma samples were histologically reviewed by a pathologist. Mutation detection was performed via a custom service by Sequenom. Cell extracts derived from human snap-frozen biopsies were processed prior to mass spectrometric analysis as previously described to analyze serine hydrolase activities. Results Mutation analysis of 40 human lung adenocarcinoma specimens identified 4 patients (10%) harbouring a cysteine for glycine substitution at position 12 (G12C) in the KRAS gene. By employing two-sided unpaired t-test we found a statistical significant (p = 0.01) activity difference of the enzyme myeloblastin between patients harbouring a G12C mutation in the KRAS gene and patients harbouring no KRAS mutation. Conclusions The results of this study revealed that the activity of myeloblastin is significantly altered in lung adenocarcinoma biopsies harboring a KRAS gene mutation. Based on the results of this study we conclude that the combination of activity-based proteomics with mutational analysis is a valid approach for the discovery of novel biomarkers for human lung adenocarcinoma.

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 4794. doi:1538-7445.AM2012-4794
Abstract 4796: Carfilzomib and ONX 0912 reduce the level of STAT3 phosphorylation in human B-cell lymphoma cells in vitro and in vivo
Authors: Jiang Jing , Kirk Christopher J. , Muchamuel Tony
Published: August 2012

BACKGROUND: Proteasome inhibitors, such as bortezomib and the tetrapeptide ketoepoxide carfilzomib (CFZ), have shown strong antitumor activity in patients with multiple myeloma and non-Hodgkin's lymphoma (NHL). Anti-tumor responses are due to direct tumor cell cytotoxicity but may also involve effects on transcription factors such as signal transducers and activators of transcription (STATs). AIM: To evaluate the effect on STAT phosphorylation in NHL by proteasome inhibitors as a possible mechanism of antitumor activity. METHODS: The human lymphoma cell lines HS-Sultan and Mino were used for this study. In vitro studies involved overnight incubation of cells with DMSO, carfilzomib or ONX 0912 by flow cytometric analysis for STAT3 and STAT5 phosophorylation. In vivo studies, utilized NIH III immunocompromised mice implanted with HS-Sultan cells and single dose administration with carfilzomib or ONX 0912 when tumors reached 200 mm3. Twenty four hours after dosing, tumor cells were isolated by enzymatic digestion and analyzed by flow cytometry. RESULTS: Carfilzomib and ONX 0912 resulted in suppressed STAT3 tyrosine phosphorylation in both cell lines in vitro and in vivo. No effects on the level of STAT5 phosphorylation were detected. CONCLUSION: These findings suggest that carfilzomib and ONX 0912 exert an anti-tumor effect against human B-cell lymphoma in part through alterations in STAT3 phosphorylation levels. This is the first report that proteasome inhibitors can reduce STAT3 activation in human B-cell lymphomas in vivo. These data support the further evaluation of carfilzomib and ONX 0912 as novel therapeutic agents for the treatment of NHL.

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 4796. doi:1538-7445.AM2012-4796
Abstract 4852: cFLIP-regulated and caspase-8-mediated limited cleavage of RIP1 promotes NF-kB activation and inhibits cell death induced by TRAIL
Authors: Habelhah Hasem , Zhang Laiqun , Janz Siegfried , Workman Lauren , Blackwell Ken
Published: August 2012

Although TRAIL is considered a potential anti-cancer agent, many types of cancer cells possess intrinsic or acquired resistance to TRAIL. In addition, TRAIL has been found to activate NF-κB and enhance metastasis in apoptosis-resistant cancer cells. Notably, caspase-8 activity and RIP1 expression have been shown to be essential for TRAIL-induced NF-κB activation. RIP1 is a dual-function molecule that can be either prosurvival or prodeath depending on the cellular context. RIP1 contains an N-terminal kinase domain (KD), a C-terminal death domain (DD) and an intermediate domain (ID) between the KD and DD domains. The ID domain of RIP1 is essential for its activation of the NF-κB pathway, whereas the KD domain is required for its induction of apoptosis and necrosis. We have discovered that, in apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID domains in response to TRAIL treatment, resulting in rapid depletion of RIP1 and the induction of apoptosis. In apoptosis-resistant cells, however, cFLIP inhibited caspase-8-mediated RIP1 cleavage in the ID but not in the KD domain, producing a KD domain-deleted C-terminal fragment, p51RIP1c. Stable expression of p51RIP1c in RIP1−/− cells significantly promoted NF-κB activation and inhibited cell death induced by TRAIL. Moreover, stable expression of the cleavage sites-mutated form of RIP1 in RIP1−/− cells, not only sensitized the cells to apoptosis induced by TRAIL, but also to necrosis induced by oxidative stress. Importantly, in Hodgkin's lymphomas that overexpress cFLIP, as well as in tumor tissues derived from iMyc-Bcl-XL double transgenic mice, a portion of RIP1 is constitutively processed to p51RIP1c. Collectively, these data suggest that cFLIP-regulated and caspase-8-mediated limited cleavage of RIP1 promotes NF-κB activation and inhibits cell death, and that such cleavage occurs constitutively in certain types of cancers. Thus, inhibiting cFLIP-regulated RIP1 cleavage could be a new target for maximizing the effectiveness of TRAIL as an anti-cancer agent.

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 4852. doi:1538-7445.AM2012-4852
Abstract 4853: Constitutively decreased TGF-β signaling and risk for colorectal cancer
Authors: Pennison Michael J. , Bellam Naresh , Zimmerman Jacquelyn , Zeng Qinghua , Sadim Maureen , Kaklamani Virginia , Li Nengjun , Zhang Kui , Stram Daniel O. , Pasche Boris
Published: August 2012

Background: Approximately one third of all colorectal cancers (CRC) are inherited, but less than 7% are attributable to known gene mutations. Germline variants within the TGF-β signaling pathway may account for colorectal cancer risk since TGF-β inhibits early tumor development through the activation of SMAD proteins. We have previously shown that Tgfbr1 haploinsufficiency is a potent modifier of colorectal cancer development in mice and humans. We have recently shown a significant association between a 3 SNP TGFBR1 haplotype and constitutively decreased TGFBR1 allelic expression, a phenotype found to occur frequently in patients with CRC. In this family-based study, we aimed at determining the association of TGFBR1 and SMAD7 with colorectal cancer risk. Methods: We studied 1,558 colorectal cancer cases and 2,640 healthy controls recruited by the Colorectal Cancer Family Registry (CCFR). We genotyped 14 TGFBR1 haplotype tagging SNPs as well as a SMAD7 SNP associated with CRC in prior GWA studies. SNPs were genotyped in all cases and controls using ABI's TaqMan platform, and associations were determined using logistical regression analysis (significance set at p<0.05). Main effects (Cockerham model) and risk prediction accuracy (receiver operating characteristic curves) were also determined. Functional assessment was conducted with derived lymphoblastoid cell lines (LCLs). Results: TGFBR1 rs7034462 and SMAD7 rs4939827 were strongly associated with colorectal cancer risk (see Table). Functional assessment performed with 3 matched pairs of patient-derived LCLs showed significantly reduced TGF-β mediated SMAD signaling in cells carrying these two SNPs. Conclusions: Our study provides the first evidence that a combination of TGFBR1 and SMAD7 variants is associated with constitutively decreased TGF-β signaling and risk for colorectal 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 4853. doi:1538-7445.AM2012-4853
Abstract 4855: KLLN, which shares a bidirectional promoter with PTEN, is an AR-regulated tumor suppressor, and induces p53-dependent and -independent apoptosis in breast cancer
Authors: Wang Yu , He Xin , Eng Charis
Published: August 2012

70% of breast cancer patients are diagnosed after menopause where the ovaries cease androgen production. Maintaining functional androgen receptor (AR) has been reported to correlate with good prognosis and better survival among breast cancer patients. We and others have shown that AR inhibits proliferation and induces apoptosis in breast cancer cells. However, the mechanism of AR's anti-tumor effect in breast cancer is still not fully understood. KLLN is a newly identified gene whose function has never been studied in tumorigenesis. KLLN and tumor suppressor gene PTEN, both located in 10q23.31, share the same promoter region but are transcribed in opposite directions. To perform in silico comparisons of KLLN expression between normal breast and breast cancer tissues, we selected 56 normal breast and 1170 tumor tissues from patients without exposure to any treatment using publicly available whole genome transcriptome datasets. We found that KLLN is significantly downregulated in breast carcinomas and that loss of KLLN expression is associated with increasing histological grade in invasive carcinomas. By using MCF-7 and MDA-MB-453 breast cancer cell lines as our models, we show that AR activates this bi-directional promoter, resulting in the upregulation of both KLLN and PTEN expression. For the first time, we identified KLLN as an AR-activated tumor suppressor, inhibiting breast cancer cell proliferation and suppressing tumor cell invasiveness in both cell lines. In MCF-7 cells, overexpression of KLLN directly activates TP53 expression as a transcription activator, accompanied by significant increase of PARP cleavage and apoptosis. We also found that KLLN could promote TP73 expression in both MCF-7 (p53-wildtype) and MDA-MB-453 (p53-null) cells at the transcriptional level, resulting in p53-independnt apoptosis. Therefore, we discovered a new pathway, which mediates AR-induced growth inhibition and apoptosis in breast cancer cells. Our study also indicates KLLN as a potential prognostic marker and novel therapy target for breast carcinomas.

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 4855. doi:1538-7445.AM2012-4855