bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3842: Methylseleninic acid, a novel potent and selective mutant-p53-rescue drug, reactivates mutant p53 by thiol modification
Authors: Qi Yanfeng , Saifudeen Zubaida R. , Morris Gilbert F. , Rowan Brian G. , Dong Yan
Published: August 2012

Mutations of the p53 tumor suppressor gene constitute the most frequent genetic alteration known in human cancers. The vast majority (∼80%) of the mutations is missense mutations, and the DNA-binding domain is the most frequently mutable region of p53. p53 mutation leads to checkpoint defects, genomic instability, and propagation of cells with genetic damage. Developing therapeutic agents that could directly bind to the mutant p53 and reconstitute p53 function has been intensively pursued and proven to be feasible. However, the challenge remains to develop a drug that specifically reactivates mutant p53 without activating the pro-apoptotic function of wild-type p53 in normal tissues. In the present study, we show that a small-molecule drug, methylseleninic acid (CH3SeO2H, MSA), can selectively reactivate mutant p53 to induce apoptosis in cancer cells. MSA can be used at a concentration at least one order of magnitude lower than that of other mutant-p53-rescue agents to achieve the same p53-reactivating and growth-inhibitory effects. MSA reduces redox-active free thiols in mutant p53 without the requirement of other proteins, potentially through forming adducts with thiols in mutant p53. The modification restores wild-type conformation to mutant p53, leading to increased DNA binding and transactivation as well as expression of growth-suppressive and pro-apoptotic target genes in both cultured cancer cells and an orthotopic tumor model. Although MSA also redox modifies recombinant wild-type p53, the modification does not lead to the induction of growth-suppressive or pro-apoptotic target genes in normal fibroblasts. Instead, the expression of p53-regulated DNA-repair genes is significantly induced in normal fibroblasts, suggesting that MSA may selectively modify p53 for DNA repair or apoptosis depending on the level of endogenous or exogenous DNA damage. Our findings, therefore, demonstrate the effectiveness and selectivity of MSA as a novel potent mutant-p53-rescue drug.

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 3842. doi:1538-7445.AM2012-3842
Abstract 3857: Smac mimetic TL32711 and TRAIL synergistically induce apoptosis of germinal center B lymphoma cells
Authors: Smith Mitchell R. , Joshi Indira , Weng David E. , Chunduru Srinivas , McKinlay Mark A.
Published: August 2012

TL32711, a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals including TRAIL (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand). TL32711 also inactivates canonical and activates non-canonical NF-κB signaling through cIAPs. We investigated the pro-apoptotic effects of TL32711, alone and in combination with TRAIL to activate the extrinsic pathway, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs activated B cell (ABC) histologies. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB dependent. We used the following EBV negative cell lines: WSU- FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 served as ABC-type DLBCL. Apoptosis was determined by annexin V staining after 48 hr incubation (TL32711 added 2-3 hr after TRAIL). The contribution of the different apoptotic pathways was determined by caspase-3 (common), -8 (extrinsic), and-9 (intrinsic) activity, assessed by flow cytometric methods and Western blot. TL32711 had little effect as a single agent on any of these B cell lymphoma cell lines at ≤ 100nM, though apoptosis was induced at 10-20 µM in GC types. TRAIL alone (10-25 ng/ml) induced apoptosis in the 3 GC lines, but had little effect on the 2 ABC lines. Combination of TL32711 and TRAIL at low concentrations showed marked apoptosis in GC lines, with minimal to no effect for each agent alone. In the combination-treated GC types, caspases -3, -8 and -9 were significantly induced, confirming activation of both pathways. We had previously shown in FSCCL and TxFL2 that caspase-8 induction also cleaved tBID, activating the intrinsic apoptotic pathway via caspase-9. GC subtypes, including FL, transformed FL and GC-DLBCL were sensitive to TRAIL and TL32711 at clinically achievable concentrations for TL32711, with supra-additive combination effects. ABC-type DLBCL were not sensitive to either agent alone or in combination. We are investigating whether ABC-DLBCL are truly dependent on canonical NF-κB activation for survival or have other mechanisms of resistance to TRAIL and IAP inhibition. These data suggest that clinical trials of TL32711 may focus on FL and GC lymphoma in combination with TRAIL or TRAIL-agonists.

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 3857. doi:1538-7445.AM2012-3857
Abstract 3883: Miniaturizing the GloSensor™ cAMP assay for MC4R screening using the Echo® acoustic liquid handler
Authors: Worzella Tracy J. , Allison Tim , Stecha Pete , Cong Mei , Zimprich Chad , Kershner Kevin , Somberg Richard L.
Published: August 2012

As biological assays used for HTS and uHTS are miniaturized, it is critical to maintain assay sensitivity while minimizing reagent volumes and maximizing throughput. The GloSensor™ cAMP Assay provides an extremely sensitive and easy to use, real-time luminescent assay format for the interrogation of over expressed or endogenous GPCRs that signal via changes in the intracellular concentration of cAMP. Tipless, touchless transfers with the Echo® liquid handler eliminate the need for costly disposable tips and greatly simplify assay development efforts. Precise and accurate drop placement eliminates cross-contamination. In this work, we demonstrate optimization of the GloSensor cAMP Assay in low-volume format using a stably transfected HEK293 cell line model expressing the melanocortin 4 receptor (MC4R) and a cAMP-sensing variant of firefly luciferase. We show that the Echo liquid handler is able to titrate small molecule and peptide agonists and antagonists, as well as transfer the GloSensor cAMP Reagent, providing robust assay results that are achieved with significantly reduced volumes of cells and compound.

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 3883. doi:1538-7445.AM2012-3883
Abstract 3885: A bi-functional fluorescent assay for the assessment of cytotoxicity and proliferation
Authors: Niles Andrew L. , Zhou Min , Bernad Laurent , McDougall Mark , Lazar Dan
Published: August 2012

Cell-based models continue to be critical tools for cancer research. Therefore, the assessment of cytotoxic or cytostatic effects remains an obligate experimental activity. Although numerous enzyme activity chemistries exist for measuring cytotoxicity, biomarker degradation may cause underestimation of actual cytotoxicity and limit the utility of these activity measures when ascertained with long-term drug exposures (72hrs or more). We have developed a cell-impermeant probe which labels the DNA from cells with compromised membranes. The quantum efficiency (i.e. brightness) of this dye increases dramatically upon DNA binding, producing a fluorescent signal which can be measured using standard “green” fluorometry wavelengths (excitation 485nm/emission 530nm). Additionally, the dye is non-toxic and stable, allowing for introduction of the probe directly into the culture medium or compound dilutions prior to cell dosing. This “no step” addition feature allows for the measurement of cytotoxicity in real time or at a convenient endpoint. Furthermore, the dye is fully compatible with luminescent measures, allowing for same-well, multi-parametric assessment of the cytotoxic phenotype. Lastly, proliferative or anti-proliferative effects (relative to control) can be revealed by the introduction of a lytic detergent which enables the normally impermeant dye to bind to all DNA in the assay well. Herein, we detail our efforts to characterize and multiplex this dye when applied to suspension and adherent cell lines treated with agents which cause either primary or secondary necrosis in a 72hr time course.

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 3885. doi:1538-7445.AM2012-3885
Abstract 3888: Next generation cell engineering: the Jump-In cell engineering platform
Authors: Vogel Kurt W.
Published: August 2012

The stable introduction of genetic material into cell lines has become one of the most widely used methods since its introduction more than 3 decades ago. Cell engineering has become an indispensable tool for a wide range of areas in Life Science, such as cell biology, physiology, In-vivo modeling, drug discovery and bioproduction. However, most cell engineering strategies currently employed for the generation of stable cell lines require substantial commitment in respect to time, FTE hours and resources. In addition, most cell lines are generated by relying on random integration of genetic material into the genome, which typically results in a wide range of expression levels within a cell population. For these reasons the frequent generation of engineered cell lines is in general cumbersome and unpredictable. Life Technologies recently developed and validated the Jump-In™ cell engineering platform, a novel gene targeting technology based on R4 integrase mediated site specific homologous recombination. This technology allows the targeted integration of genetic material into a specific pre-engineered site, which by design reduces the effort required for generation of stable cell lines compared to standard methods. Isogenic expression from a defined genomic locus provides the ideal solution for comparative analysis of gene families, isotypes or orthologs. This technology was applied to two widely used cellular backgrounds, HEK293 and CHO-K1. We used these cellular backgrounds as case studies to demonstrate how this technology can be applied to rapidly generate engineered cell lines for a variety of cell based assay applications, including reporter assays for pathway analysis, GPCR activity assays and analysis of protein post-translational modifications. In summary, Jump-In™ technology enables researchers to accelerate the development of cellular assays by greatly reducing the amount of time and resources required for the upfront cell engineering process. For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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 3888. doi:1538-7445.AM2012-3888
Abstract 3895: Rational design of small molecule inhibitors that bind to an allosteric pocket on human heat shock protein 70 (Hsp70)
Authors: Taldone Tony , Patel Pallav D. , Kang Yanlong , Rodina Anna , Talele Tanaji T. , Chiosis Gabriela
Published: August 2012

The 70 kDa heat shock protein (Hsp70) plays an important role in cancer and its pharmacological modulation with small molecules may represent a useful therapeutic approach. The discovery of such agents has been hampered by the lack of a full length crystal structure of human Hsp70. We constructed a homology model of the human Hsp70, which we investigated for potential druggable sites using SiteMap tool. An allosteric site in the nucleotide binding domain was identified and used to design potent Hsp70 modulators. Analysis of the Glide docking resulted in an understanding of important interactions between designed ligands, such as YK5, and the Hsp70 protein. When tested in breast cancer cells, YK5 led to depletion of onco-proteins, induced apoptosis and inhibited growth. YK5 is to our knowledge the first rationally designed small molecule inhibitor of Hsp70, and thus, represents a novel chemical tool to investigate its potential in cancer and other diseases.

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 3895. doi:1538-7445.AM2012-3895
Abstract 3910: Lead optimization of a series of 5-aminopyrazol-imidazopyridine compounds as potent anaplastic lymphoma kinase inhibitors active against clinically relevant ALK mutations
Authors: Yang Bin , Saeh Jamal C. , Peng Bo , Kamhi Victor , Harrison Rane , Harish Reema , Wu Allan , Whitston David , Wang Haiyun , Ye Minwei , Wang Suping , Code Erin , Rivard Caroline , Drew Lisa , Mazzola Anne Marie , Russell Daniel J.
Published: August 2012

Despite the success of Crizotinib in treating NSCLC patients with EML4-ALK fusions, cancers eventually develop resistance via a variety of mechanisms including mutations in the ATP binding site of the kinase domain. A series of 5-aminopyrazol-imidazopyridine compounds were identified to potently inhibit anaplastic lymphoma kinase and were found to be active against a number of mutations in vitro but suffered from strong Cyp inhibition and were found to be metabolically unstable by incubation in human hepatocytes and microsomes. Installation of a pyrimary alcohol group on the heteroaryl ethyl group of the scaffold consistently improved Cyp3A4 liability, and led to improvement in physical and DMPK properties as well as improvement of their metabolic stability in human Heps. The lead compound from this series was orally administrated to SCID mice in a Del xenograft model and achieved greater than 90% phospho-ALK inhibition over 6 hours post dose at 10 mg/kg dose. The lead compound was subsequently tested against Crizotinib-resistant ALK mutations in enzyme assays, and was shown to inhibit the clinically relevant ALK mutations, including L1196M. In enzyme assays, it was inactive against G1269S mutation, consistent with modeling studies. Subsequent profiling in an FDCP cell line over expressing ALK G1269S mutation showed potent anti-proliferative activity, suggesting off-target activity. Further characterization of this series identified AurB inhibition to be a key cell cycle kinase responsible for the off-target activity in the engineered cell line. The off target activity hinders it from assessing the primary pharmacology responsible for its preclinical efficacy in disease relevant models. In conclusion, we have identified a novel orally bioavailable compound that is a potent dual active ALK and AurB inhibitor that is active against clinically-relevant gatekeeper mutant.

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 3910. doi:1538-7445.AM2012-3910
Abstract 3913: Estrogen receptor beta and estrogen receptor beta 5 in triple negative breast cancer cells: IGF-II role in regulation of estrogen receptor beta 5
Authors: O'Bannon Chane’
Published: August 2012

African American (AA) women with breast cancer are more likely to be diagnosed at a more advanced stage of the disease, have a higher recurrence rate and a poorer prognosis than Caucasian (CA) women. They also are more often diagnosed with high-grade breast tumors at an earlier age (pre-menopausal), most of which are negative for the estrogen and progesterone receptors (ER-, PR-). These factors result in a higher mortality rate among AA breast cancer patients. Our lab has demonstrated that AA women have increased levels of IGF-II coupled with lower levels of the IGF-II receptor needed to degrade IGF-II, thus leading to higher IGF-Ii signaling in contrast to CA women. AA women also have a higher expression of the insulin receptor A versus insulin receptor B when compared to CA women, which increases proliferation and decreases metabolic effects (Singh et. al 2011). We have also shown that IGF-II activates the estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-α) in the absence of the estrogen ligand (Richardson et. Al 2011). When co-expressed, ER-α counteracts the proliferative function of ER-α, however the role of ER-α in breast cancer is different in ER-α negative compared to ER-α positive tumors. Furthermore, there are 5 different isoforms of ER-α, of which ER-Δ5 is the most abundantly expressed form in AA women. Since IGF-II activates ER-α and ER-Δ5 is associated with increased mortality and decreased time recurrence, we have designed a study to determine if IGF-II regulates ER-Δ5. We hypothesize that in the absence of ER-α, ER-α may activate the ER-α signaling pathway, and that the elevated expression of ER-Δ5 and its heterodimerization with of ER-α1 contributes to the overall survival disparity. To test this hypothesis we characterized the levels of phophorylated ER-α (pER-α) and compared them to IGF-II levels in triple negative breast cancer cells and normal triple negative epithelial cells. IGF-II levels were analyzed using western blotting for protein, and RT-PCR for mRNA. AA women have a higher expression of IGF-II in both malignant and normal cells, and comparably higher levels of expression of the active form of ER-α when compared to CA breast cancer cell lines. IGF-II stimulation promoted the translocation of ER-Δ5 to different cellular compartments. We propose that ER-Δ5 impairs the inhibitory effects of ER-α and thus may contribute to the survival disparity among AA breast cancer 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 3913. doi:1538-7445.AM2012-3913
Abstract 3922: 20(S)-protopanaxadiol-aglycone inhibition of the development and growth of castration-resistant prostate cancer
Authors: Cao Bo , Liu Xichun , Xiong Zhenggang , Zhao Lijuan , Zhang Haitao , Dong Yan
Published: August 2012

Recurrence with lethal castration-resistant prostate cancer after androgen deprivation therapy remains the major challenge in treatment of advanced prostate cancer. The main mechanism for the development of castration resistance is continuous androgen receptor signaling via promiscuous or hypersensitive androgen receptor, ligand-independent androgen receptor activation, expression of androgen receptor splice variants lacking the ligand-binding domain but remaining constitutively active, and/or de novo androgen synthesis in the prostate. Several new drugs targeting androgen receptor reactivation, such as abiraterone acetate, are showing benefit in prolonging survival in patients with castration-resistant prostate cancer. However, even this potent inhibitor of androgen synthesis has a relatively short time to disease progression. One potential mechanism is the prevalent upregulation of constitutively active, ligand-binding-domain-truncated androgen receptor splice variants in castration-resistant prostate cancer. Therefore, therapeutic approaches that can diminish the availability of both the full-length and the splice variants of androgen receptor should offer considerable benefit in preventing and inhibiting prostate cancer recurrence after androgen deprivation therapy. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD), a steroidal saponin from ginseng, effectively downregulates the abundance and activity of both the full-length and the splice variants of androgen receptor. The effect is manifested by an immediate drop in androgen receptor proteins, followed by a reduction in androgen receptor mRNAs. The initial decrease in androgen receptor proteins could be attributed to PPD induction of proteasome-mediated degradation, possibly as a result of disrupted androgen receptor N-C interaction. Our xenograft studies show that PPD inhibits not only the development of castration-resistant prostate cancer after androgen deprivation but also the growth of prostate tumors that are already castration resistant. The inhibition is accompanied by a pronounced decrease of tumor androgen receptor and serum prostate-specific antigen levels. Our findings therefore provide strong justification and lay the groundwork for an effective treatment modality to improve the therapeutic outcome of androgen deprivation therapy.

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 3922. doi:1538-7445.AM2012-3922
Abstract 3935: Microfluidic analysis of triple negative breast cancer epithelial cells in response to chemotherapeutic treatments coupled with oxygen gradients
Authors: Byrd Thomas F. , Seale Kevin
Published: August 2012

Multi-trap nanophysiometers (MTNPs) are used in trapping and studying very small numbers of cells. These precisely manufactured instruments, made of polydimethylsiloxane (PDMS), contain microfluidic channels and chambers that house arrays of three-sided cell traps. Trapped MCF7 breast cancer cells were perfused with media and gradients of chemotherapeutic agents specific to triple-negative breast cancer while oxygen levels were modulated within the device and quantified by the incorporation of an oxygen-sensing film. Rapid switching between perfusion channels and gas enabled precise dosing of cells with chemotherapeutic agents in a chamber simulating a variety of tumor microenvironments ranging from anoxic to normoxic. Preliminary results show a positive correlation between oxygen concentration and non-translational motion of trapped cells over a four hour time period.

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 3935. doi:1538-7445.AM2012-3935
Abstract 3939: Hypoxia-induced BRCA1 phosphorylation and degradation
Authors: Lu Yuhong , Glazer Peter Michael
Published: August 2012

Hypoxia is a key feature of tumor microenviroment. We have demonstrated that hypoxia downregulates BRCA1 at the transcriptional level through promoter binding by E2F4/p130 complexes and that hypoxia can produce BRCA1 gene silencing via epigenetic modifications. We also demonstrated that downregulation of BRCA1 by hypoxia leads to decreased DNA repair capacity, establishing a mechanism by which hypoxia can drive genetic instability in cancer cells. In addition to transcriptional regulation, BRCA1 has been found to be phosphorylated at S988 in a Chk2-dependent manner by both ionizing radiation and hypoxia exposure. In this study, we have been able to confirm that other residues of BRCA1 are phosphoylated during hypoxia exposure, including S1387, S1423, S1466, and S1524. Intriguingly, the phosphoryation of BRCA1 only happens under severe hypoxic condition, such as 0.01% oxygen, but not under moderate hypoxia (1% oxygen condition). Furthermore, the BRCA1 phosphoylation events induced by hypoxia can be attenuated by an ATM inhibitor, KU55933, indicating that ATM is playing a role in hypoxia-induced BRCA1 phosphorylation at multiple sites. We also observed that the decrease in BRCA1 protein levels in hypoxia is partially blocked by MG132, a proteasome inhibitor. Consistent with this, by transient transfection of exogenous BRCA1, we found that hypoxia induces BRCA1 unbiquitination in MCF-7 cells. Together, our data suggest that hypoxia-induced downregulation of BRCA1 is in part mediated through the ubiqutin-proteasome pathway. Our findings indicate a new mechanism of hypoxia induced regulation of BRCA1 by modulating its stability, suggesting that hypoxia-induced downregulation of BRCA1 is through multiple mechanisms. Future studies will investigate the mechanism of hypoxia-induced BRCA1 phophorylation and the relation between hypoxia-induced BRCA1 phosphorylation and BRCA1 degradation. To understand more about regulation of BRCA1 by hypoxia could lead to new therapies to target hypoxic tumors.

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 3939. doi:1538-7445.AM2012-3939
Abstract 3946: Genome-wide characterization of hypoxic breast cancer transcriptome
Authors: Choudhry Hani , Oikonomopoulos Spyros , Harris Adrian , Ragoussis Ioannis
Published: August 2012

Hypoxia is a common feature of most solid tumors and is associated with poor prognosis in breast cancer. In the current study, we investigated the hypoxia transcription landscape of an ER positive breast cancer cell line [MCF7] using high throughput strand-specific total RNA sequencing (tssRNA-seq). MCF7 cells were grown under hypoxia and normoxia (24 hours, 1% O2), and we found about 7.3% of annotated genome got significantly differential expressed in hypoxic stress, which includes 79% (1293) up regulated and 21% (344) down regulated transcripts (P value of <0.01, fold >1.5). Analysis revealed deregulation of a range of non coding transcripts including piwiRNA (0.36%), miRNA (0.098%), sn/snoRNA (0.028%), tRNA (4.5%), lncRNA (6.5%) under hypoxia (P value of <0.01, fold >1.5). Many non-poly A transcripts and natural anti-sense transcripts (n=71) were detected and differently expressed in hypoxia. We characterized two significant up regulated natural anti-sense transcripts in hypoxia and found that these transcripts are long (>200bp), lack of protein coding frames, localized in nucleus and found highly expressed (range fold >5-15) in different hypoxic breast cancer cell lines. To define which hypoxic altered transcripts are dependent on, hypoxia-inducible factor (HIF), we suppressed HIF-alpha subunits in hypoxic MFC-7 cells using short interfering RNA and subjected them to mRNA-seq. Results revealed that HIF-1, HIF-2 and combined HIF-1/2 knockdown resulted in significant down-regulation of 179, 84 and 149 transcripts, respectively, which were up regulated in hypoxic MCF-7 cells. Despite detection of previously reported HIF dependent transcripts, we detected a substantial number of HIF dependent transcripts (n=64) that were not reported before as hypoxia regulated genes. To our understanding, this is first study to show hypoxia dependent natural anti-sense transcripts and genome-wide HIF dependent transcripts analysis at single base pair resolution in hypoxic tumors. These finding will open new avenues in our efforts to understand the hypoxia induced transcriptional response.

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 3946. doi:1538-7445.AM2012-3946
Abstract 3952: New advances in regulation of senescence by PML and the PML nuclear bodies
Authors: Aauino Mariana D. Acevedo , Bourdeau Véronique , Vernier Mathieu , Ferbeyre Gerardo
Published: August 2012

Senescence is a cellular defense mechanism activated by short telomeres, DNA damage or expression of oncogenes. The program includes the expression of high levels of the promyelocytic leukemia protein PML that form nuclear spherical bodies, known as PML-nuclear bodies (PML-NB). The expression of PML in normal fibroblasts is sufficient to induce senescence while genetic inactivation of PML inhibits the process. These results suggest that PML is a critical component of the senescence tumor suppressor mechanism. Accordingly PML is poorly expressed in malignant human tumors but highly expressed in benign tumors. We recently discovered a new mechanism of regulation of RAS-induced senescence by PML, which implicates the recruitment of the RB/E2F complex to the PML-NB via RB-PML interaction. This leads to inhibition of cell cycle and DNA repair genes, DNA damage, p53 activation and ultimately to senescence. We show now that the cyclin dependent kinase CDK4 blocks the ability of PML to regulate E2F gene expression and senescence and that CDK inhibitors potentiate the ability of PML to restore the senescence program in tumor 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 3952. doi:1538-7445.AM2012-3952
Abstract 3956: BRCA1 chromatin dissociation induced by oncogene activation promotes formation of senescence-associated heterochromatin foci and senescence via chromatin remodeling factor BRG1
Authors: Tu Zhigang , Zhang Rugang
Published: August 2012

Background: Cellular senescence is a state of irreversible cell growth arrest that is an important tumor suppression mechanism in vivo. Activation of oncogenes such as Ras in primary mammalian cells typically triggers senescence and formation of senescence-associated heterochromatin foci (SAHF) that contributes to senescence-associated cell cycle exit. We have previously showed that dissociation of BRCA1 from chromatin induced by oncogenic Ras plays a key role in promoting cellular senescence by driving SAHF formation. However, the mechanism by which BRCA1 chromatin dissociation promotes SAHF formation and senescence remains to be determined. BRG1 is an ATP dependent SWI/SNF chromatin-remodeling factor that interacts with BRCA1. Components of SWI/SNF complex including BRG1 are often deregulated in human cancer. Here we examined the role of BRG1 in regulating formation of SAHF trigged by BRCA1 chromatin dissociation induced by oncogenic Ras. Results: We discovered that the interaction between BRG1 and BRCA1 is disrupted during oncogenic RAS induced senescence. Interestingly, BRG1 accumulates in chromatin fraction while BRCA1 becomes dissociated from chromatin. Notably, BRG1 knockdown suppresses formation of SAHF and senescence, while has no effects on BRCA1 chromatin dissociation in Ras-infected cells, suggesting that BRG1 functions downstream of BRCA1 chromatin dissociation in promoting SAHF formation and senescence. Indeed, BRG1 knockdown is sufficient to inhibit SAHF formation and senescence induced by BRCA1 knockdown. Conversely, BRG1 overexpression drives SAHF formation and senescence. Notably, senescence induced by BRG1 overexpression is independent of DNA damage response, further supporting the idea that BRG1 functions downstream of BRCA1 chromatin dissociation during Ras induced senescence. Mechanistically, chromatin immunoprecipitation analysis revealed that, in Ras-expressing or BRCA1 knockdown cells, there is an increased association of BRG1 with the promoters of p16INK4a and p21CIP, both of which are key regulators of SAHF formation and senescence. Significance: These studies revealed the molecular underpinning by which BRCA1 chromatin dissociation promotes SAHF formation and senescence. They also imply that BRG1 inactivation contributes to cancer development by suppressing cellular senescence. (R.Z. is an Ovarian Cancer Research Fund (OCRF) Liz Tilberis Scholar. This work was supported in part by a NCI FCCC-UPenn ovarian cancer SPORE (P50 CA083638) pilot project and SPORE career development award, a DOD ovarian cancer academy award (OC093420), and an OCRF program project.)

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 3956. doi:1538-7445.AM2012-3956
Abstract 396: Sequential metastases of colorectal cancer: Treatment, immunophenotypes and spatial distributions of infiltrating immune cells
Authors: Halama Niels , Keim Sophia , Zoernig Inka , Schirmacher Peter , Grabe Niels , Jaeger Dirk
Published: August 2012

The role of the immune system in the course of colorectal cancer has been elucidated in the last decade. While quantification of the immune cell infiltrates within the the resected specimen at diagnosis has a clear power to estimate the prognosis of the patient, the role within the metastatic situation and especially within the metastatic lesion itself remains largely unclear. Recent analyses of infiltrates in colorectal cancer liver metastases revealed a role for the infiltrate density not only for prognosis but also in the prediction of treatment response. This not only broadens the view on these infiltrates and indicates a systematic role of the local immunological microenvironment, but also raises the question how these infiltrates change during repeated courses of treatment (i.e. resection, chemotherapy etc.). To adress this question, multiple sequential lung or sequential liver metastases of colorectal cancer patients were analyzed using whole slide image quantification, using antibodies against CD3, CD8, FOXP3, CD68 and CD163. The clinical data and interventions were analyzed in association with the individual patient and the profiles of the metastatic lesions. The resulting cell densities reveal a heterogeneous profile: after successfull treatment of a metastatic lesion, the recurrent lesion can still have the same immunophenotype with similar cell distributions. In a situation of a favorable immune cell profile, this profile can return and apparently convey a similar favorable course of the disease. But also the opposite was found: the recurrent metastatic lesion could have a different profile with alterations in specific immune cell subsets. Treatment failure and a progression were observed after this change in immune cell composition. Further analyses are required to elucidate the different patterns and their associations to the treatment, the tumor cell phenotype and other factors. It is clear from this data however, that there is an immune cell plasticity during the course of the disease that needs to be analyzed and maybe has potential for improving therapeutic decisions.

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 396. doi:1538-7445.AM2012-396
Abstract 3960: Rapid and efficient large-scale somatic mutation validation approaches for cancer genome sequencing
Authors: Cibulskis Kristian , Sivachenko Andrey , Hammerman Peter , Imaz Ivan , Pugh Trevor , Sougnez Carrie , Winckler Wendy , Gabriel Stacey , Getz Gaddy
Published: August 2012

The landscape of somatic alterations in cancer is being rapidly uncovered by large-scale cancer genome sequencing projects in individual laboratories as well as consortia such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). The decreasing cost of sequencing has resulted in an increase in the number of cancer genomes that are being sequenced, and thus the numbers of candidate somatic mutations being identified are rapidly expanding. A single cancer genome from a cancer with an average mutation background rate, such as ovarian or breast cancer, will have 3000-6000 mutations. A high mutation rate cancer such as melanoma or colon cancer can have as many as 100,000 mutations/genome. Multiplying these numbers by the dozens of sequenced genomes and hundreds of sequenced exomes across a wide spectrum of tumor types yields a staggering number of candidate mutations. However, approaches for rapidly validating these findings at scale have been lagging behind. Current approaches, such as custom designed hybrid capture arrays followed by sequencing, can take months to complete and cannot begin until the initial sequencing is complete and mutations are called. This can lead to long delays in the interpretation and publication of biological findings. Here we describe the results of several approaches for rapid and efficient validation on large-scale projects, such as TCGA. We will share our approach and results using Fluidigm and PacBio sequencing for validation and sample extension at a small scale with rapid turnaround where we have validated PIK3CA and TP53 mutations in breast cancer samples of varying purity as well as significantly mutated genes in medulloblastoma. Ideally one could validate mutations and gene fusions concurrent with their discovery. To that end, we will also describe our validation results using RNA-Seq data, commonly produced concurrently with genome and exome sequencing, in lung squamous and renal cancer. Finally, we will explore an approach for instant-validation through simultaneous sequencing of barcoded discovery and cross-validation 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 3960. doi:1538-7445.AM2012-3960
Abstract 3967: The Cancer Genome Project high throughput analysis pipeline
Authors: Butler Adam P. , Teague Jon W. , Raine Keiran M. , Menzies Andrew , Jones David , Marshall John , Hinton Jon , Dronov Serge , Gamble John , Stebbings Lucy , Jayakumar Alagu , Leroy Catherine , McDermott Ultan , Stratton Michael R. , Campbell Peter , Futreal Andy
Published: August 2012

The Cancer Genome Project (CGP) was set up in 2000 to use systematic mutation screening methods to increase our understanding of human cancer. With the advent of next generation sequencing, and the large volumes of data that it generates, a new suite of software was required to rapidly and accurately screen these data for somatic changes. We have built an analysis pipeline to track and analyse large numbers of tumour samples, using in-house and externally available tools. The analysis pipeline is built around a ∼2,000 node compute farm and Lustre filesystem which outputs into our archive and data storage system, FileTrk. FileTrk holds the raw data files (BAM, CEL etc), the results of the analysis and any versioning information about the software used to generate these results. Sample lanes are aligned back to the genome using Burrows-Wheeler Aligner (BWA) and lane-to-lane comparisons are made to ensure data integrity. Lanes from each sample are merged into a single sample BAM file and once 30 - 40x coverage is reached and the lanes have been quality assessed the sample is locked and ready for analysis. Mutation callers detect point mutations (Caveman, in-house software), small insertions/deletions (Pindel), breakpoints (BRASS, in-house software) and copy number changes (ASCAT & PICNIC, in-house software). The resulting mutations are post-processed to remove false positives, annotated to the RNA and protein level using standard nomenclature (Vagrent, in-house software) and uploaded to a database. Interfaces have been developed to enable the selection of random sets of mutations for validation, the outcome of the validations is recorded so specificity can be calculated for each sample in the system. IT systems are being developed to automatically export lists of somatic changes to COSMIC, the ICGC data portal and raw data to the European Genome-Phenome Archive (EGA).

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 3967. doi:1538-7445.AM2012-3967
Abstract 3968: Exploring cancer datasets in the integrative genomics viewer (IGV)
Authors: Robinson James T. , Thorvaldsdottir Helga , Mesirov Jill
Published: August 2012

Cancer genome characterization studies, such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), are producing a flood of diverse data including whole-genome sequencing scans, expression profiles, high-resolution SNP and copy number data, and epigenetic profiles. These diverse datasets are enabling researchers to study the cancer genome in unprecedented detail. However, their sheer size and diversity presents significant challenges to downstream interpretation. While much progress has been made in automated analysis, human review enabled by intuitive and responsive visualizations remains an essential component of data analysis. The Integrative Genomics Viewer (IGV) was developed to address this need by providing researchers with an intuitive, user-friendly, interactive visualization tool for exploration of diverse genomic datasets. IGV has extensive support for both next-generation and micro-array based platforms, and for integration of these data types with clinical and phenotypic data. It provides an intuitive interface similar to online mapping tools such as Google Maps, enabling smooth zooming and panning at all resolution scales, from whole genome to base pairs. Data can be annotated, filtered, grouped, and sorted in a variety of ways. This ability to dynamically and flexibly integrate multiple different datasets, and view them at any scale, allows investigators to elucidate complex biological relationships that are not otherwise readily apparent. In this presentation we describe the IGV, with emphasis on recent features focused on data integration and interpretation, developed in close collaboration with cancer researchers. Specifically (1) a multi-locus pathway view which supports simultaneous viewing of data in multiple genomic regions defined by pathways or gene sets, (2) integration with external tools such as Mutation Assessor and PolyPhen, to provide views and data to asses the functional significance of somatic events, and (3) a flexible, interactive charting capability to enable detailed exploration of inter-dependencies between data types at a specific locus or set of loci. These features will be illustrated in the context of use cases drawn from the TCGA glioblastoma multiforme and ovarian datasets.

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 3968. doi:1538-7445.AM2012-3968
Abstract 397: Soluble Tumor Necrosis Factor Receptor shed by breast tumor cells modulates macrophage migration
Authors: Rego Stephen , Ricci Krista , Swamydas Muthulekha , Dreau Didier
Published: August 2012

Tumor necrosis factor alpha (TNFα) present within the tumor microenvironment, especially in breast tumor with a strong inflammatory component, promotes tumor death. TNFα also stimulates the migration of immune cells including macrophages to the tumor site. Breast cancer cells express both TNFR1 and TNFR2, the cell surface receptors for TNFα. The breast tumor microenvironment also contains soluble forms of TNFR1 and TNFR2. We investigated the origins and effects of soluble TNFR1 and TNFR2 on monocyte/macrophage cell migration. Our results indicate that both sTNFR1 and sTNFR2 were shedded by tumor cells but also by stroma cells including adipocytes. In addition, the amounts of sTNFR2 shedded by adipocytes and 4T1 mammary tumor cells were significantly higher than the amounts of sTNFR2 shedded by normal murine epithelial NMuMG cells (p<0.05). The migration of M2 subtype macrophages but not of M1 subtype macrophages derived from either murine bone derived monocytes, J774 or Raw cells toward 4T1 mammary tumor cells’ conditioned media was significantly increased following treatment of 4T1 cells with the sheddase inhibitor TAPI-0 (p<0.05). Indeed, TAPI-0 treatment decreased sTNFR2 concentrations in 4T1-conditioned media (p<0.05). These observations suggest a role for sTNFR1 and sTNFR2 in the dynamic interplays between immune and tumor cells during breast progression. (This works is supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007)

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 397. doi:1538-7445.AM2012-397
Abstract 4249: Development of Champions Tumorgraft models in rare cancer types for preclinical investigation and drug discovery
Authors: Friedman Jay A. , Kilonzo Christine , Albee Lee , James Meredith , Lu Bin , Bruckheimer Elizabeth
Published: August 2012

Little progress has been achieved in the treatment of the rare cancers such as Ewing Sarcoma (ES), Merkel cell carcinoma (MCC), and osteosarcoma. ES is a rare bone malignancy that affects children and young adults and there has been little improvement in survival rates for patients with metastatic or recurrent disease despite aggressive treatment. MCC is a rare malignant cutaneous tumor of the elderly and optimal treatment strategies remains unclear for these patients. Poor clinical outcome is seen especially those MCC patients with regional lymph node metastases and distant metastases. Osteosarcoma arises in bone and affects adolescents and young adults. There has been no significant improvement in prognosis since the advent of multi-agent chemotherapy. A significant limitation in sorting out the complexities of these rare tumors and evaluating therapeutic strategies is the lack of preclinical models. To overcome this challenge, we have embarked on a mission to establish and characterize Champions TumorgraftTM models within these rare tumor indications. The Champions Tumorgraft utilizes the implantation of primary human tumors in immune-deficient mice in a manner that preserves the biological properties of the original human tumor. In this preliminary work, we reveal several new Champion TumorgraftTM models of ES, MCC, and osteosarcoma along with their tumor histology, mutational status, and chemosensitivity profiles to several standard of care compounds. These rare Champion TumorgraftTM models represent invaluable tools for preclinical research and drug discovery to find promising new therapies for this high unmet clinical need.

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 4249. doi:1538-7445.AM2012-4249