bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4417: Targeting Ras with small molecules that activate SOS-mediated nucleotide exchange
Authors: Rossanese Olivia W. , Burns Michael C. , Sun Qi , Daniels R. Nathan , Camper DeMarco V. , Kennedy J. Phillip , Phan Jason , Olejniczak Edward T. , Lee Taekyu , Waterson Alex G. , Fesik Stephen W.
Published: May 2015

Aberrant activation of Ras, by mutation or constitutively active upstream kinases, occurs in nearly 30% of all human cancers, rendering Ras one of the most validated targets in cancer drug discovery. Historically, the direct inhibition of Ras with small molecules has proven extremely difficult. Here we report the discovery of compounds that bind to a unique pocket on the Ras:SOS:Ras complex, which increase the rate of SOScat-catalyzed nucleotide exchange in vitro and modulate Ras signaling pathways in cells. X-ray crystallography reveals that the molecules bind in a hydrophobic pocket in the CDC25 domain of SOS, adjacent to the Switch II region of Ras. Structure-based mutational analyses confirmed the functional relevance of this binding site and demonstrated that it is essential for compound activity. Consistent with their increased exchange activity in vitro, these molecules increase Ras-GTP levels in cells. However, the compounds inhibit ERK and AKT phosphorylation; the ERK inhibition at high concentrations is accompanied by an increase of p-ERK levels at lower compound concentrations. These molecules represent a unique tool to study the acute activation of Ras and highlight a novel pocket on SOS that may be exploited to modulate Ras signaling.

Citation Format: Olivia W. Rossanese, Michael C. Burns, Qi Sun, R. Nathan Daniels, DeMarco V. Camper, J. Phillip Kennedy, Jason Phan, Edward T. Olejniczak, Taekyu Lee, Alex G. Waterson, Stephen W. Fesik. Targeting Ras with small molecules that activate SOS-mediated nucleotide exchange. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4417. doi:10.1158/1538-7445.AM2014-4417
Abstract 4423: Somatic mutations in the insulin-like growth factor-1 and insulin receptors found in cancer genomes result in constitutive activation of the receptor kinases
Authors: Greer Ann , Hillerman Stephen , Chang Han , Wong Tai Wai
Published: May 2015

Somatic mutations in receptor tyrosine kinases have been shown to be a mechanism by which tumor cells subvert signaling pathways to drive deregulated cell proliferation. The insulin-like growth factor 1 receptor (IGF-1R) and the related insulin receptor (InsR) regulate cell metabolism and cell proliferation by phosphorylating two related adapter proteins called insulin receptor substrates (IRS1/2). The IRS proteins are unique to the IGF-1R/InsR signaling pathway, and are recruited to the receptors by virtue of the interaction between the SH2 sequences of IRS1/2 and tyrosine auto-phosphorylation sites on the receptors. The complete sequence determination of cancer genomes has now made it possible to assess the extent of mutation in components of the IGF-1R/InsR signaling pathway. Analyses of The Cancer Genome Atlas (TCGA) data from colorectal cancer patients revealed that >25% of the tumor type have copy number gain in the genes encoding IRS2 or the ligand IGF2. These observations suggest a role for the IGF-1R/InsR signaling pathway in the pathobiology of colorectal cancer. To better understand the extent to which the IGF-1 and insulin receptors may be oncogenic drivers, we surveyed the TCGA data sets for somatic mutations in IGF-1R and InsR. To date, over 50 non-synonymous missense mutations in each of the two receptors have been identified across multiple tumor types. Mutations occur in both the extracellular and intracellular sequences, and few mutations are recurrent. cDNA containing IGF-1R and InsR mutations were analyzed for their intrinsic kinase activities. A number of mutations in the kinase domains resulted in constitutive activation of the receptor kinases, in a similar manner to what has been observed with other receptor tyrosine kinases. Effects of these mutations on the ability of the receptors to drive signal transduction and cell proliferation have been analyzed and will be reported. Results of these studies have further validated a role for the IGF-1R and InsR signaling pathway in cancer.

Citation Format: Ann Greer, Stephen Hillerman, Han Chang, Tai Wai Wong. Somatic mutations in the insulin-like growth factor-1 and insulin receptors found in cancer genomes result in constitutive activation of the receptor kinases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4423. doi:10.1158/1538-7445.AM2014-4423
Abstract 4429: Loss of function Cbl-c mutations in solid tumors
Authors: Kales Stephen C. , Ryan Philip E. , Nau Marion M. , Green Jeffrey E. , Lipkowitz Stanley
Published: May 2015

Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and disruption of this regulation can lead to tumor initiation and progression. Cbl proteins are a family of RING finger ubiquitin ligases that negatively regulate a variety of RTKs, including EGFR, MET, RET and components of the TCR pathway. Recent studies have identified a growing number of Cbl mutations associated with human myeloid neoplasias and epithelial tumors. Cbl-c is the most recently identified human Cbl protein and is expressed exclusively in epithelial cells. Recently, a novel cDNA has been isolated from the ductal carcinoma of a mouse mammary cancer model. This mutant has a deletion in the catalytic domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeoloid neoplasias. Genomic analysis of both parental and transgenic lines shows no evidence of germline mutation indicating that this mutation is likely a somatic mutation. In overexpression studies, this mutant cDNA fails to mediate ubiquitination and degradation of activated EGFR and, acting as a dominant negative, enhances cell transformation. Furthermore, data mining reveals several Cbl family mutations associated with solid tumors in humans. Subsequent cell-based analysis demonstrates a similar loss of E3 function in some of these human mutations. Through subsequent analysis of transforming capacity and signal pathway regulation, we are assessing interacting partners of Cbl-c and the potential role of these mutations in tumorigenesis.

Citation Format: Stephen C. Kales, Philip E. Ryan, Marion M. Nau, Jeffrey E. Green, Stanley Lipkowitz. Loss of function Cbl-c mutations in solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4429. doi:10.1158/1538-7445.AM2014-4429
Abstract 4440: TRAF4 is a key molecule for lung cancer through regulating AKT ubiquitination and activation
Authors: Peng Cong , Li Wei , Chen Xiang , Dong Zigang
Published: May 2015

TRAF4 is a member of TRAF family. Although TRAF4 was reported to be overexpressed in cancer tissues, the role of TRAF4 in tumorigenesis remains unclear. Here, we found TRAF4 is over expressed in lung cancer cell lines as well as lung cancer tissues. Knockdown TRAF4 dramatically attenuated lung cancer malignant phenotype including cell proliferation, anchorage independent growth and tumor development in a xenograft mouse model . To our surprised, it is that TRAF4, but not skp2 is required for Akt activation through ubiquitination in lung cancer. Overall, our study suggests that TRAF4 might be a potential molecular target for lung cancer prevention and therapy.

Citation Format: Cong Peng, Wei Li, Xiang Chen, Zigang Dong. TRAF4 is a key molecule for lung cancer through regulating AKT ubiquitination and activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4440. doi:10.1158/1538-7445.AM2014-4440
Abstract 4483: IO125, a novel Pt-based supramolecular therapeutic exhibits increased anti-cancer efficacy compared with oxaliplatin
Authors: Roy Monideepa , Hossain Sk Samad , Sarkar Arindam , Sengupta Aniruddha , Gupta Nimish , Hussain Sajid , Ansari Aasif , Mylavarapu Sanghamitra , Sengupta Shiladitya
Published: May 2015

Despite the widespread use of platinum chemotherapy, only few drugs have been approved in this class of molecules. Here, we describe a novel supramolecular chemistry-based platform technology to engineer more potent oxaliplatin analogues. In IO125, a unique platinum (II) is tethered to a cholesterol backbone via a monocarboxylato bond and an N→Pt coordination environment, releasing diaminocyclohexane (DACH)-platinum in a sustained pH-dependent manner. The molecule facilitates assembly into defined nanoscale supramolecular structures of uniform size distribution as measured using dynamic light scatter and electron microscopy. Such a supramolecular therapeutic demonstrates enhanced activity compared with oxaliplatin in 4T1 and MDA-MB-231 triple negative breast cancer lines. The uptake of IO125-based supramolecular therapeutic was 10-20 fold greater than free oxaliplatin in these cells. In addition, the nanoparticles exhibited significantly lower IC50 values in comparison to oxaliplatin in primary colorectal carcinoma lines generated from patient tumor sample. Nanostructures have been shown to exhibit superior pharmacokinetics profile and biodistribution to tumors due to an enhanced permeability and retention (EPR) effect. Based on these observations, we anticipate an IO125-based supramolecular therapeutic may emerge as a novel next-generation platinum-based potent anti-cancer agent.

Citation Format: Monideepa Roy, Sk Samad Hossain, Arindam Sarkar, Aniruddha Sengupta, Nimish Gupta, Sajid Hussain, Aasif Ansari, Sanghamitra Mylavarapu, Shiladitya Sengupta. IO125, a novel Pt-based supramolecular therapeutic exhibits increased anti-cancer efficacy compared with oxaliplatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2014-4483
Abstract 4490: A novel tumor-targeting construct aimed at c-Met
Authors: mastali mitra , Sims Jessica D , Taguiam Jan M , Hanson Chris , Valenteen Felix Alonso , Medina-Kauwe Lali K.
Published: May 2015

C-Met is a receptor tyrosine kinase (RTK) that is activated by the binding of its ligand, hepatocyte growth factor (HGF). The HGF/c-Met pathway contributes to embryonic development, wound healing, cell proliferation and survival, motility and morphogenesis. It is also one of the most frequently dysregulated pathways in a variety of human cancers, including breast, ovarian, prostate, lung, and pancreatic. Importantly, cell surface elevation of c-Met has been associated with drug-resistance, including acquired resistance to current signal-blocking therapies.

Tumor-targeting strategies that do not require signal inhibition may prove more effective on c-Met positive cancer cells. This may be addressed by ligands that recognize c-Met to trigger cell uptake of attached therapeutics, thus bypassing the need to block signaling. HGF has the potential to accomplish this but its requirement for tetramerization and disulfide bonding presents technical complications for therapeutics development. An alternative, and potentially superior ligand for c-Met targeting may possibly be derived from a bacterium that causes food-poisoning.

The human pathogen, Listeria monocytogenes, binds c-Met to invade host cells through its surface proteins called Internalins. Specifically, Internalin B (InlB) triggers receptor-mediated endocytosis after c-Met binding. InlB and HGF recognize different regions of c-Met, and InlB does not require tetramerization or disulfide bonds for binding. Consequently, InlB is more suitable for recombinant protein production than HGF.

We have previously shown that PBK10, a recombinant protein derived from the adenovirus capsid penton base, can mediate gene and drug delivery into cells through the membrane penetrating activity of the penton base. Moreover, PBK10 can be targeted to tumor cells via recombinant fusion to tumor-homing ligands. Here, we explore the possibility of fusing the receptor-binding site of InlB to PBK10 for targeting attached cytotoxic agents to c-Met positive cancer cells, and transporting such agents directly into these cells via receptor-mediated endocytosis and membrane penetration.

Citation Format: mitra mastali, Jessica D Sims, Jan M Taguiam, Chris Hanson, Felix Alonso Valenteen, Lali K. Medina-Kauwe. A novel tumor-targeting construct aimed at c-Met. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4490. doi:10.1158/1538-7445.AM2014-4490
Abstract 4550: Differential PARP inhibitors' effects on cell cycle and consequently homologous DNA damage repair
Authors: Jelinic Petar , Levine Douglas A.
Published: May 2015

Background: Poly (ADP)-ribose polymerase (PARP) inhibition has been explored in pre-clinical and clinical studies with greatest successes demonstrated in homologous DNA damage repair (HDR) pathway-deficient cancer patients. PARP inhibitor's (PARPis) potency to inhibit PARP's catalytic activity is well established; however non-catalytic effects that may add to their therapeutic properties are not fully understood. Our objective was to comparatively evaluate non-catalytic effects of olaparib and veliparib, the two most widely used PARPis in clinical trials, with a particular focus on cell cycle effects and HDR.

Methods: To measure DNA damage repair activity upon PARPi treatment, we used a well-established DR-GFP reporter assay in two model cell lines, U2OSDR-GFP and H1299DR-GFP. Cell cycle progression was assessed by propidium iodide and monitored by flow cytometry. Depletion of targeted mRNAs was performed by standard siRNA knock-down technique using electroporation for transfection. Immunoblotting was used to confirm cell cycle results and demonstrate replicative stress on the protein expression levels.

Results: We found a dramatic decrease in DNA damage repair activity in olaparib- versus veliparib-treated U2OSDR-GFP p53 wild type cells, regardless of inhibitory potency. This decrease in HDR activity was a result of olaparib triggering a cell cycle arrest-like effect as a response to strong replicative stress. Lack of the same effect in p53-null cells suggests that olaparib's effect is p53-dependent, which was confirmed in U2OSDR-GFP cells depleted of p53. Finally, CHK1 is strongly phosphorylated in cells treated with olaparib but not veliparib, confirming cells differential response to these two PARPis.

Conclusion: These data demonstrate that olaparib and veliparib differ in their non-catalytic inhibitory effects, with olaparib but not veliparib mitigating DNA damage repair activity via cell cycle arrest-like effects in a p53-WT background. Moreover, olaparib but not veliparib triggers CHK1 phosphorylation, which suggests differential levels of replicative stress. This new insight into olaparib's and veliparib's mechanism of action should be considered when developing inclusion criteria for treatment of cancer patients with these PARP inhibitors.

Citation Format: Petar Jelinic, Douglas A. Levine. Differential PARP inhibitors' effects on cell cycle and consequently homologous DNA damage repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4550. doi:10.1158/1538-7445.AM2014-4550
Abstract 457: Expression, purification, and characterization of recombinant TAT-SALL4B protein
Authors: Yuan Mengru , Jiang Yongping , Dai Wei
Published: May 2015

SALL4 is an important transcription factor that supports expansion of hematopoietic stem cells. Several recent studies reveal that SALL4 and SALL4B (a major isoform) are heavily modified by post-translational mechanisms and that these modifications are critical for their stability, subcellular localization, and transcriptional activities. Given the importance of SALL4 and SAL4B in supporting stem cell self-renewal and expansion, we optimized a large scale expression and purification process to obtain SALL4B. Sf9 insect cells were transduced with the recombinant baculovirus expressing human SALL4B fused with 6His residues and a TAT sequence in the N-terminus. Recombinant TAT-SALL4B was purified by nickel affinity chromatography under native conditions. Immuno-blotting confirmed that recombinant SALL4B was highly expressed and purified. As the first step to test the biological activity of purified TAT-SALL4B, we investigated whether TAT-SALL4B, directly supplemented to the culture medium, was capable of entering into cells through the protein transduction process. Immuno-fluorescence microscopy revealed that recombinant TAT-SALL4B specifically localized to the nucleus in a concentration- and time-dependent manner. To further test the transcriptional activity of TAT-SALL4B protein, dual luciferase reporter assay was performed, which showed that TAT-SALL4B protein was transcriptionally active.Our results suggest that TAT-SALL4B may provide a promising cellular factor for supporting ex vivo expansion of hematopoietic stem cells.

Citation Format: Mengru Yuan, Yongping Jiang, Wei Dai. Expression, purification, and characterization of recombinant TAT-SALL4B protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 457. doi:10.1158/1538-7445.AM2014-457
Abstract 4982: Characterization of novel α-ketoamide derivatives as potent inhibitors against cathepsin S induced by acidic extracellular pH in pancreatic cancer cells
Authors: Chang Wun-Shaing Wayne , Chang Tzu-Yuan , Wu Tzu-Chin , Liao Chien-Yu , Lin Chun-Cheng
Published: May 2015

Extracellular acidification is known to be a driving force in pancreatic cancer growth and metastasis. Thus, manipulation of acidic peritumoral pH or blockage of key proteins stimulated by acidic extracellular microenvironment may offer considerable potential for pancreatic cancer therapy. Cathepsin S, also known as CTSS or CatS, is a critical proteolytic enzyme found to be up-regulated in malignant cells and secreted into the extracellular milieu to degrade surrounding matrix components. By examining the effect of low pH (6.7) on various pancreatic tumor cell lines, we detected a consistently increased CTSS expression associated with augmented cell migration and invasion. Other features such as CTSS-mediated proteolysis and ECM degradation were also observed under mildly acidic condition. Based on these findings, we designed and synthesized some new small molecules bearing an α-ketoamide warhead to evaluate their ability to inhibit CTSS. Kinetic study revealed these compound inhibitors possess very low Ki values and high specificity against target CTSS enzyme. Further in vitro and in vivo analyses demonstrated these agents not only could protect fibronectin from CTSS-mediated degradation but also induce tumor cell autophagy under extracellular acidification. Together these results indicate the potential of α-ketoamide derivatives as antitumor agents against pancreatic cancer.

Citation Format: Wun-Shaing Wayne Chang, Tzu-Yuan Chang, Tzu-Chin Wu, Chien-Yu Liao, Chun-Cheng Lin. Characterization of novel α-ketoamide derivatives as potent inhibitors against cathepsin S induced by acidic extracellular pH in pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2014-4982
Abstract 5037: Targeting MICA with therapeutic antibodies for the treatment of cancer
Authors: Blery Mathieu , Bonnafous Cécile , Peri Valentine , Trichard Sylvia , Perrot Ivan , Cornen Stéphanie , Thielens Ariane , Breso Violette , Morel Yannis , Romagne François , Rossi Benjamin , Paturel Carine , Gauthier Laurent
Published: May 2015

MICA and MICB, along with ULPBs, are ligands for the activating receptor NKG2D expressed on NK cells and subsets of T cells in Human. NKG2D ligands are induced by cellular stress and pathogen infections. Their expression is tightly regulated by complex mechanisms both at the mRNA and protein levels. In the case of MICA and MICB, more than 65 and 30 alleles respectively were described with different properties regarding to their cellular location adding to the complexity of this recognition system. Nevertheless, as markers of cellular stress, in particular in tumorigenesis, MICA and the closely related MICB proteins are candidates of choice to be targeted by a cytotoxic therapeutic antibody. We first evaluated MICA/B expression by immunohistochemistry on healthy tissues and tumors to validate these antigens as therapeutic targets. Then, using mouse immunization, we generated a panel of chimeric human IgG1 monoclonal antibodies targeting MICA and MICB. These mAbs have the ability to bind to several structurally different alleles and to cross-react on MIC proteins from cynomolgus macaques. Their capacity to block the MICA/NKG2D interaction was assessed by surface plasmon resonance as well as by using cell-based assays. In vitro efficacy was measured by the capacity to mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC) towards MICA expressing cells. In vivo efficacy of the anti-MICA mAbs was measured in both a preventive and a curative setting using MICA expressing cell lines. Altogether, we have generated a panel of anti-MICA mAbs with diverse functional properties. Ongoing work aims to choose the best candidate for humanization and further clinical development.

Citation Format: Mathieu Blery, Cécile Bonnafous, Valentine Peri, Sylvia Trichard, Ivan Perrot, Stéphanie Cornen, Ariane Thielens, Violette Breso, Yannis Morel, François Romagne, Benjamin Rossi, Carine Paturel, Laurent Gauthier. Targeting MICA with therapeutic antibodies for the treatment of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5037. doi:10.1158/1538-7445.AM2014-5037
Abstract 5099: Automated rapid and accurate cell cycle analysis
Authors: Yurlova Larisa , Gregor Jacqueline , Sirenko Oksana , Cromwell Evan , Zolghadr Kourosh
Published: May 2015

It is essential to have detailed information about compounds identified in screening campaigns during drug discovery and to know their influence on cell cycle progression or DNA repair. The discovery of Chromobodies® and advancements in High Content Imaging systems with improved optical properties and live cell capabilities has opened the door for new reliable and robust screening assays that provide these relevant high quality data.

Chromobodies are a novel class of fluorescent antibodies, based on special heavy chain antibodies from camelids (VHHs). These unique nanoprobes can be transfected into cells as plasmid DNA similar to any conventional fusion protein. The cell expresses the fluorescent antibody-fragment thus enabling the non-invasive imaging of endogenous proteins and processes in real time. Therefore Chromobodies avoid any deleterious side effects from the traditional over-expression of a tagged protein. This is especially true for the essential components of the DNA replication, DNA repair and DNA methylation machinery: Proliferating Cell Nuclear Antigen (PCNA) and DNA Methyltransferase 1 (Dnmt1) . It has been shown that high over-expression of theses proteins can have adverse effects on normal progression through S phase.

In this study we present a new method to evaluate cell cycle and DNA methylation. We developed Chromobodies directed against PCNA and Dnmt1. Transfecting these Chromobodies into cells circumvents the laborious and error-prone staining procedure with conventional antibodies. We demonstrate the flexible use of the Cell Cycle Chromobody cell line in conjunction with different automated high content imaging platforms. We show dose-dependent cell cycle inhibition caused by a number of anti-cancer drugs, DNA damage and apoptosis inducing compounds. Multiple analysis protocols enable the fast and accurate analysis with quantitative information on each sub phase of DNA replication for each individual cell. Moreover, by continuing to film cells, the exact length the cell cycle stages can also be extrapolated, thereby providing detailed temporal information on G1, S, G2 and M phase.

Citation Format: Larisa Yurlova, Jacqueline Gregor, Oksana Sirenko, Evan Cromwell, Kourosh Zolghadr. Automated rapid and accurate cell cycle analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5099. doi:10.1158/1538-7445.AM2014-5099
Abstract 5219: MicroRNA-874 impedes angiogenesis in non-small cell lung cancer
Authors: Chetty Chandramu , Maddirela Dilip R. , Gogineni Venkateswara R. , Chittivelu Subramanyam , Rao Jasti S. , Moorthy Bhagavatula
Published: May 2015

MicroRNAs (miRs) are short (20-24nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in eukaryotes by affecting both the stability and translation of mRNAs. MicroRNAs play crucial roles in normal biological processes and are commonly dysregulated in human diseases. Thus far, miRs have been shown to act as either tumor suppressor genes or oncogenes in driving critical gene expression pathways in cancer stem cells in a wide range of human malignancies, including non-small cell lung cancer (NSCLC).We previously found that miR-874 could suppress tumor growth and metastasis of NSCLC. Since angiogenesis is important for tumor growth and metastasis, in this study, we further investigated the possible roles of miR-874 in tumor angiogenesis. Attenuation of miR-874 expression in histological sections of NSCLC tumors and the CD133 positive population of NSCLC cells (NSCLC-initiating cells; NSCLC-ICs), as compared to its normal counterparts, was observed by in situ hybridization and real time quantitative PCR, respectively. Tumor-conditioned medium from NSCLC-ICs induced significantly high levels of endothelial cell capillary network and neovascularization in vivo; whereas, re-expression of miR-874 in NSCLC-ICs inhibited this capillary network formation ability by suppressing the protein levels of VEGF, Stat3, MMP-2, uPA and PI3K. Further, miR-874-treatment decreased in vivo angiogenesis and pre-established orthotopic tumor growth in nude mice as compared to mock- and control-miR-treatments. Immunohistochemical analysis revealed a significant decrease in the expression of VEGF, Stat3 and microvessel density (CD31 levels) in xenograft tumor sections from miR-874-treated mice. In conclusion, our study highlights the possible anti-angiogenic role of miR-874 in NSCLC-initiating cells and presents a new, hopeful therapeutic target and prognostic marker for NSCLC.

Citation Format: Chandramu Chetty, Dilip R. Maddirela, Venkateswara R. Gogineni, Subramanyam Chittivelu, Jasti S. Rao, Bhagavatula Moorthy. MicroRNA-874 impedes angiogenesis in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5219. doi:10.1158/1538-7445.AM2014-5219
Abstract 5539: Therapeutic efficacy of novel selenium-containing HDACi in acute leukemia
Authors: Sachdev Mansi , Desai Dhimant , Muthusami Sunil , Gowda Chandrika , Song Chunhua , Amin Shantu G. , Dovat Sinisa
Published: May 2015

Despite tremendous advances in treatment of leukemia that have resulted in increased survival of patients with this disease, therapy for high-risk and relapsed leukemia remains a clinical challenge. This necessitates the development of novel, more potent therapeutic modalities. Epigenetic therapy with various types of inhibitors of histone deacetylases (HDACi), have demonstrated therapeutic efficacy in a range of malignancies, often with minimal side effects. Dr. Shantu Amin's group recently reported the development of a potent, novel, selenium-containing HDAC inhibitor (SelSA-1), which showed efficacy against Hodgkin's lymphoma and chronic myelogenous leukemia cells, as well as against lung cancer and melanoma cell lines. Here we expand efficacy studies of selenium-containing HDAC inhibitors to include a broad range of hematopoietic malignancies and begin to address the drug effects on cellular functions. We tested the therapeutic efficacy of two novel HDAC inhibitors - SelSa-1 and SelSa-2 on acute lymphoblastic leukemia, acute myelogenous leukemia and non-Hodgkin's B cell lymphoma. Cell proliferation assays demonstrated sharp dose-dependent cytotoxic effects in all the above cell lines at 96-hours post-treatment. Quantitative analysis showed that SelSa-1 and SelSa-2 have distinct effects in different types of hematopoietic malignancies. In T-cell acute lymphoblastic leukemia (T-ALL) and in non-Hodgkin's lymphoma, both SelSa-1 and Selsa-2 have similar therapeutic activity with IC50 = 1uM and 1-2uM, respectively. In B-cell acute lymphoblastic leukemia, SelSa-1 has superior therapeutic activity compared to SelSa-2 with IC50 = 0.2 uM vs. 1.25, respectively. In acute promyelocytic leukemia (APML) without the t:15:17 translocation, SelSa-1 was superior to SelSa-2 (IC50= 0.25uM and 1uM respectively), while APML with the t:15:17 translocation showed similar sensitivity to both compounds with an IC50 of 1-2uM. Cell cycle analysis performed by flow cytometry revealed that these agents induce G1-cell cycle arrest in leukemia cells. Preliminary data suggest that both SelSa-1 and SelSa-2 have a synergistic effect with standard chemotherapeutic agents that induce DNA-damage. In summary, these results demonstrate the effectiveness of two selenium-containing analogs of SAHA, SelSA-1 and 2, on leukemia cells from multiple hematopoietic lineages. Additional studies to identify detailed mechanisms of the effects of these drugs on cellular function are underway.

Citation Format: Mansi Sachdev, Dhimant Desai, Sunil Muthusami, Chandrika Gowda, Chunhua Song, Shantu G. Amin, Sinisa Dovat. Therapeutic efficacy of novel selenium-containing HDACi in acute leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5539. doi:10.1158/1538-7445.AM2014-5539
Abstract 555: DNA methylation patterns are associated with subpopulations of diffuse large B-cell lymphoma
Authors: Wu Jenny , McCord Ron , Sandmann Thomas , Walter Kim , Bourgon Richard , Soriano Robert , Modrusan Zora , Darbonne Walter , Mundt Kirsten E.
Published: May 2015

Diffuse large B-cell lymphoma (DLBCL) comprises two predominant molecular subtypes: germinal-center B-cell-like (GCB) and activated B-cell-like (ABC). These subtypes were initially identified by their gene expression profiles, which are reminiscent of the cell of origin of the lymphoma. Though histologically indistinguishable, these subtypes are associated with distinct clinical outcomes and may be predictive for response to novel therapies. The development of improved diagnostic biomarkers for these DLBCL subtypes could therefore inform clinical decision-making by enabling selection of patients likely to respond to such novel therapies.

To investigate regulatory mechanisms potentially responsible for the differential gene expression patterns seen in the DLBCL subtypes and to identify potential biomarkers for these subtypes, we performed whole genome methylation profiling of 15 DLBCL cell lines and 21 DLBCL patient tissues and identified distinct DNA methylation patterns associated with the two DLBCL molecular subtypes.

Analysis of the methylation patterns of several DLBCL classifier genes known to be differentially expressed in ABC compared to GCB revealed the classic DNA methylation correlation pattern: high mRNA expression was associated with low methylation in the vicinity of the transcriptional start site (TSS), and vice versa. Furthermore, comparison of differential methylation profiles with corresponding RNA-seq data further supports the notion that DNA methylation profiles underlie the gene expression patterns that are currently used to classify ABC and GCB DLBCL subtypes. Collectively, these data suggest that DNA methylation may contribute to the regulation of expression of key ABC and GCB DLBCL classifier genes and further, that the DNA methylation state at these loci could potentially serve as useful diagnostic biomarkers for the DLBCL subtypes.

Citation Format: Jenny Wu, Ron McCord, Thomas Sandmann, Kim Walter, Richard Bourgon, Robert Soriano, Zora Modrusan, Walter Darbonne, Kirsten E. Mundt. DNA methylation patterns are associated with subpopulations of diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 555. doi:10.1158/1538-7445.AM2014-555
Abstract 5573: p62/IMP2 promotes breast cancer cell progression
Authors: Li Yang , Peng Bo , Ning Jing , Qian Wei , Francia Gulio , Zhang Jianying
Published: May 2015

Breast cancer is one of the major causes of cancer-related death for women throughout the world. Although the early detection of breast cancer is improving, the strategies available to treat the advanced stages of this disease remain limited. Therefore, the identification of key molecules that regulate the metastatic process is necessary to provide the rationale for further therapeutic interventions. The IGF2 mRNA-binding Protein 2 (IMP2/IGF2BP2/p62) is a member of the IMP family. Expression of IMPs in cancer cells is associated with cancer progression. Our previous studies showed that p62/IMP2 is overexpressed in breast cancer tissue compared to the adjacent normal breast tissue. However, the role of p62/IMP2 in breast cancer progression remains unknown. Here, we present evidence that transfection of p62/IMP2 into two p62/IMP2 negative cell lines (MDA-MB-231 and MCF7) increases 5 to 10 folds (P < 0.05) in-vitro wound healing and reduces 2 to 3 folds (P < 0.05) cell adhesion to the extracellular matrix. Unexpectedly, the overexpression of p62/IMP2 did not affect cell proliferation in our breast cancer cell lines.

Our analysis shows that the overexpression of p62/IMP2 leads to increased c-myc expression in MDA-MB-231 cells but not MCF-7 cells at both the transcriptional and translational levels. In contrast, beta-catenin expression is increased by p62/IMP2 only in MCF7 cells. Since c-myc and beta-catenin gene products are important regulators of metastasis in breast cancer, and their increased expression is observed in our p62/IMP2 transfectants, our data suggests that p62/IMP2 may be an important mediator of breast cancer progression, although its mechanism of action may vary depending on the cellular context in which it is expressed.

Citation Format: Yang Li, Bo Peng, Jing Ning, Wei Qian, Gulio Francia, Jianying Zhang. p62/IMP2 promotes breast cancer cell progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5573. doi:10.1158/1538-7445.AM2014-5573
Abstract 5589: Molecular diagnostic, prognostic and therapeutic role of mir-221 in pancreatic cancer
Authors: Sethi Seema , Sarkar Shaan , Dubaybo Hala , Ali Shadan , Goncalves Priscila , Kollepara Spilakshmi , Philip Philip , Li Yiwei
Published: May 2015

Background: Comprehensive molecular profiling of microRNAs (miRNAs) and subsequent target gene analysis of pancreatic cancer (PaCa) can provide tumor specific miRNAs signatures to improve diagnostic accuracy, refine prognostic and predictive capabilities, and may also serve as therapeutic targets. In PaCa such a comprehensive analysis has not been reported.Design: RNA extracted from scant amounts of formalin fixed paraffin embedded PaCa tumor and normal pancreatic tissues were profiled for miRNA expression using microfluidic biochip microarrays (which interrogate 2019 unique mature miRNAs,LC Sciences). The expression of abnormal miRNAs was then validated and quantified using real time RT-PCR. Data was statistically analyzed using the Student's t-test. Survival data obtained in each case was correlated with the miRNA data using Kaplan-Meier analysis. Oncogenic potential, therapeutic modulation and downstream target genes of altered miRNA were evaluated by real time RT-PCR using PaCa cells for mRNA expression.Results: miRNA profiling showed high expression of miR-221 in PaCa tissues compared to normal pancreas (p=0.001). These findings were validated and quantified using qRT- PCR (p=0.0029) . Survival analysis using Kaplan-Meier, demonstrated shorter survival of patients with higher expression of miR-221 compared to those with relatively lower levels of miR-221 . The cell proliferation index of PaCa cells was increased by miR-221 mimics transfection and decreased by miR-221 inhibitors. The predicted target genes of miR-221 identified by RT-PCR were PTEN, p27kip1, p57kip2 and PUMA.Conclusions: High throughput miRNA expression profiling showed high expression of miR-221 in PaCa tissues. Higher levels of miR-221 demonstrated poorer prognosis, suggesting the oncogenic potential of miR-221 in PaCa. Its target genes are PTEN, p27kip1, p57kip2 and PUMA which are tumor suppressors, and found to be deregulated by over-expression or under-expression of miR-221 transfection studies in PaCa cells. These results provide molecular evidence of oncogenic potential of miR-221 in PaCa which may have a significant clinical impact on prognosis & risk stratification and in designing novel targeted molecular therapeutics in the future to achieve the goal of personalized and precision medicine.

Citation Format: Seema Sethi, Shaan Sarkar, Hala Dubaybo, Shadan Ali, Priscila Goncalves, Spilakshmi Kollepara, Philip Philip, Yiwei Li. Molecular diagnostic, prognostic and therapeutic role of mir-221 in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5589. doi:10.1158/1538-7445.AM2014-5589
Abstract 5606: Detection of circulating tumor DNA in early and late stage human malignancies
Authors: Bettegowda Chetan , Sausen Mark , Leary Rebecca , Kinde Isaac , Agrawal Nishant , Bartlett Bjarne , Wang Hao , Luber Brandon , Kinzler Kenneth , Vogelstein Bert , Papadopoulos Nickolas , Diaz Luis
Published: May 2015

The development of minimally-invasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital PCR-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We initially use targeted, exomic, or whole genome sequencing to identify sequence or structural alterations in tumor tissues of 410 patients using targeted, exomic, or whole genome sequencing. We found that at least one tumor-specific mutant molecule could be identified in <5 mL of plasma in >75% of patients with advanced ovarian, colorectal, bladder, gastroesophoageal, pancreatic, breast, melanoma, hepatocellular and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73%, 57%, 48% and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. Circulating tumor DNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% while and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor (EGFR) blockade in 24 patients who objectively responded to therapy but who subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase (MAPK) pathway. Remarkably, nearly half (45%) of the observed mutations observed were in codon 61 of either the KRAS or NRAS gene. Taken together, these data suggest that ctDNA is a broadly applicable, sensitive, specific and robust biomarker that can be used for a variety of clinical and research purposes in patients with several multiple different types of cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Chetan Bettegowda, Mark Sausen, Rebecca Leary, Isaac Kinde, Nishant Agrawal, Bjarne Bartlett, Hao Wang, Brandon Luber, Kenneth Kinzler, Bert Vogelstein, Nickolas Papadopoulos, Luis Diaz. Detection of circulating tumor DNA in early and late stage human malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5606. doi:10.1158/1538-7445.AM2014-5606
Abstract 562: Impact of ERα expression status and ESR1 genetic variation on progression in tamoxifen-treated breast cancer patients
Authors: Babyshkina Nataliya , Vtorushin Sergey , Patalyak Stanislav , Dronova Tatyana , Slonimskaya Elena , Cherdyntseva Nadejda
Published: May 2015

Background. Resistance to tamoxifen remains a major clinical challenge in the treatment of breast cancer. The aim of this study was to evaluate the influence of the intensity and the pattern of estrogen receptor α (ERα) expression and common single nucleotide polymorphisms (SNP) in the estrogen receptor 1 gene (ESR1, rs2228480:2014G>A; rs2077647:+30T>C) on disease progression in breast cancer patients who received adjuvant tamoxifen.

Material and methods. Formalin-fixed paraffin-embedded tumor tissue samples were retrospectively collected from 61 patients, who had distant metastasis versus distant metastasis-free breast cancers (n = 37 versus n = 24, respectively, with a 9-year follow-up). Protein expression of ER, PR and HER2 were assessed by immunohistochemistry. Real-time polymerase chain reaction analysis was used to determine the ESR1 SNPs.

Results. We found that the heterogeneous distribution of ERα was statistically significant related with a low expression level and intensity of ERα in tumor tissue samples (p=0.002 and p=0.0002, respectively). In addition, the level and intensity of ERα expression was lower in patients with distant metastasis compared to distant metastasis-free cases, but these differences were not significant (P>0.05). Nonetheless, the ESR1 2014A allele was associated with a heterogeneous distribution of ERα expression (p=0.02). We showed the higher risk of distant metastasis development for the patients carrying the ESR1 2014A mutant allele compared with women with ESR1 2014G variant (p=0.04).

Conclusion. These data suggest that the expression level and intensity of ERα with ESR1 2014G>A genetic variation could be a useful molecular markers for predicting tamoxifen treatment prognosis in breast cancer patients.

Citation Format: Nataliya Babyshkina, Sergey Vtorushin, Stanislav Patalyak, Tatyana Dronova, Elena Slonimskaya, Nadejda Cherdyntseva. Impact of ERα expression status and ESR1 genetic variation on progression in tamoxifen-treated breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 562. doi:10.1158/1538-7445.AM2014-562
Abstract 565: Telomerase reverse transcriptase promoter alterations in human bladder cancer
Authors: Turan Sevilay
Published: May 2015

The telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of the enzyme telomerase, (aka telomere terminal transferase), which contains both protein and RNA subunits. TERT is a RNA-dependent polymerase which increases the length of telomeres at the ends of each chromosome by adding TTAGGG repeats. TERT polymerase uses RNA as a complementary primer to elongate telomeric G-rich repeat sequences in the 5’ to 3’ direction. TERT is essential and active in germ cells, stem cells, primary tumors, and immortalized cells and acts to maintain adequate telomeres. However, in somatic cells, TERT expression is reduced or absent and telomeres gradually shorten during the S phase of each cell cycle due to replication-dependent loss of telomere terminal sequences. Adequate telomere length is important for preserving genome stability, regulating cell replication, and preventing cell death. Somatic TERT promoter alterations were recently reported in melanoma, glioma, and in small sample sets from other cancers including bladder cancer. These are thought to drive re-expression of TERT in cancer cells. In this study, given recent reports of somatic TERT promoter alterations in bladder cancer, polymerase chain reaction and Sanger sequencing were performed to examine a region of the TERT promoter in 58 bladder tumors. The TERT promoter was altered in 90% of 58 tumors by 44 germline (21 unique, 20 novel) and 53 somatic variants (11 unique, 4 novel). Somatic alterations were observed in 67% of tumors. The majority of variants were C>T DNA changes, three variants occurred as both germline and somatic, and a subset of variants created or altered transcription factor binding sites and promoter CpGs. Analyses of telomere length showed significantly shorter telomeres in tumor tissue versus adjacent normal tissue from the same patient (p=0.004) indicating TERT may not sufficiently lengthen telomeres in rapidly proliferating cancer cells. Interestingly, somatic TERT promoter alterations were not associated with stage, grade, or alterations in other bladder cancer genes identified by exome sequencing. We speculate that TERT promoter alterations are frequent and early events in bladder cancer and identification of TERT alterations in urine may provide a biomarker for early diagnosis and monitoring of bladder cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Sevilay Turan. Telomerase reverse transcriptase promoter alterations in human bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 565. doi:10.1158/1538-7445.AM2014-565