bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4235: Site specific profiling of histone methyltransferases in cancer cells using histone peptide microarray containing a comprehensive set of histone peptides.
Authors: Zhu Bing , Hong Ailing , Hebel Chris , Zhou Xiaochuan , Gao Xiaolian
Published: August 2014

Post-translational modifications (PTMs) of histones play a critical role in diverse biological processes including chromatin compaction, gene expression and cell differentiation. Among a myriad of PMTs, histone methylation catalyzed by histone methyltransferases (HMTs) has been increasingly recognized as an important player responsible for a major signaling mechanism in eukaryotic cells. This suite of epigenetic modifiers represents a new and promising class of therapeutic targets. In cancer, there is a growing body of evidence that suggests changes in the activity of HMTs (a class of chromatin-modifying enzymes) contribute to the uncontrolled cell proliferation that is a hallmark of this devastating disease. The sequence specificity of the substrates of HMTs under a cellular condition are largely unknown but known targets have been mostly identified through a conventional candidate-based approach by using purified HMTs. However, such an experiment frequently does not reflect what could be occurring in cellular contexts or in vivo. In this study, we designed and synthesized a comprehensive histone peptide microarray (PepArray) on a microfluidic chip which contains 3,919 peptides. The peptides contain nine residues with the methylation sites and mutant sites situated in the middle of the sequence so that each peptide has a unique possibility for modification such as methylation or acetylation. We obtained nuclear extract from the breast cancer cell line T47D, and applied the protein lysates to the histone methylation PepArray chip. After incubation of the chip with a methyl-specific antibody, significant signals were detected at the sites containing peptides corresponding to H2AK74, H3K122, and H4K59. We found null signal at mutant sites where the target lysine(K) was replaced with alanine(A). These results reveal the specific activity profiles of HMTs at defined histone sites in a cellular system. Planned further investigation will compare the different histone methylation or acetylation profiles in the various cellular systems, especially in different cancer systems. The current experiment demonstrates an effective solution to comprehensive studies of epigenetic modification. This information may be translated into therapeutic targets of histone methylation inhibition by focusing on identifying inhibitors of specific HMTs as targets for a new generation of therapeutics.

Citation Format: Bing Zhu, Ailing Hong, Chris Hebel, Xiaochuan Zhou, Xiaolian Gao. Site specific profiling of histone methyltransferases in cancer cells using histone peptide microarray containing a comprehensive set of histone peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2013-4235
Abstract 4236: Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro.
Authors: Goueli Said A. , Hsiao Kevin , Zegzouti Hicham
Published: August 2014

Methylation/demethylation of DNA and Proteins play major roles in modulation of the epigenome and has been implicated in a wide variety of human diseases. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. The assay is universal since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, protein, and small molecules methyltransferases. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background and low C.V. The assay is robust (Z’ value > 0.7) and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates with no limitations on the use of high concentrations of substrates or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest.

Citation Format: Said A. Goueli, Kevin Hsiao, Hicham Zegzouti. Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2013-4236
Abstract 4241: Isozyme-selective intracellular binding assay for histone deacetylases .
Authors: Woodroofe Carolyn C. , Robers Matthew B. , Kirkland Thomas A. , Kupcho Kevin , Niles Andrew L. , Cong Mei , Wood Keith V.
Published: August 2014

The precise modulation of histone acetylation is crucial to cellular survival and control of reproduction.Misregulation of acetylation is often correlated with uncontrolled cellular proliferation, and general inhibition of histone deacetylases has proven to be an effective strategy in the treatment of some cancers. The underlying biology of histone deacetylases is however quite complex, with four classes of isozymes and a large number of interacting proteins that modulate the activity of each. The general scarcity of potent isozyme-selective HDAC inhibitors further complicates the task of deciphering individual isozyme activity in a cellular context. We now report a novel approach to monitoring the intracellular inhibition of single isozymes based on bioluminescence resonance energy transfer from NanoLucTM, a small and exceedingly bright bioluminescent reporter. In our approach, a NanoLucTM fusion of the isozyme of interest is used in combination with an optimized fluorescent tracer. When bound, energy transfer occurs from the bioluminescent enzyme to the fluorescent probe. Displacement of the probe by a competitive inhibitor is then measured as a decrease in the energy transfer ratio, yielding IC50 data specific to the chosen isozyme. We demonstrate the ability to measure intracellular IC50 values of inhibitors against individual isozymes of class I and class IIb based on choice of the NanoLucTM fusion.

Citation Format: Carolyn C. Woodroofe, Matthew B. Robers, Thomas A. Kirkland, Kevin Kupcho, Andrew L. Niles, Mei Cong, Keith V. Wood. Isozyme-selective intracellular binding assay for histone deacetylases . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4241. doi:10.1158/1538-7445.AM2013-4241
Abstract 4274: Prolidase directly binds and activates epidermal growth factor receptor and stimulates downstream signaling.
Authors: Yang Lu
Published: August 2014

Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxy terminus. Unexpectedly, we have found that recombinant human PEPD directly binds and activates epidermal growth factor receptor (EGFR), and that EGFR activation by PEPD in turn leads to activation of multiple signaling molecules downstream of EGFR, including but may not be limited to AKT, ERK and STAT3. EGFR is a cell surface receptor that plays a major role in a variety of cellular responses, and EGFR activation is known to result in increase in DNA synthesis, cell growth, cell proliferation and cell migration, which perhaps are most clearly demonstrated in cancer cells. The ability of PEPD to activate EGFR signaling is neither cell-specific nor dependent on its enzymatic activity; in the latter case, an enzymatically inactive PEPD mutant was similarly effective in activating EGFR, AKT, ERK and STAT3. PEPD is also a novel inducer of cyclooxygenase-2 (Cox-2), known to play an important role in cancer, inflammation and other diseases, but Cox-2 induction by PEPD also depends on EGFR activation. In line with the pro-survival and pro-proliferation activities of EGFR and Cox-2, PEPD stimulates cell proliferation and DNA synthesis in cultured cells. However, PEPD could activate EGFR only when it was present in the extracellular space, as forced expression of human PEPD via transient gene transfection failed to activate EGFR and the downstream signaling proteins. This result is nonetheless consistent with EGFR being a cell surface receptor. Although PEPD is normally a cytosolic protein, it is apparently released from injured cells and tissues in vivo, and such release appears to result in EGFR activation and activation of its downstream signals. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor. Indeed, there are several notable differences between PEPD and EGF with regard to binding and activation of EGFR and activation of downstream signaling molecules. In conclusion, PEPD is a new ligand of EGFR and presents a novel mechanism of EGFR activation.

Citation Format: Lu Yang. Prolidase directly binds and activates epidermal growth factor receptor and stimulates downstream signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4274. doi:10.1158/1538-7445.AM2013-4274
Abstract 4275: Production and characterization of ErbB1 (EGFR) associated with nanolipoprotein particles via cell-based and cell-free methods.
Authors: Scharadin Tiffany M. , Takanishi Christina , He Wei , Saldano Matthew , Carraway Kermit L. , Coleman Matthew A. , Henderson Paul T.
Published: August 2014

The four members of the ErbB family of mammalian receptor tyrosine kinases are essential for the development and maintenance of a variety of tissues. Aberrant activation or over-expression of these receptors leads to the progression of epithelial and brain tumors. An understanding of the mechanistic and structural changes that occur to activate the intracellular receptor tyrosine kinase domain via extracellular ligand binding and receptor dimerization is desirable for identifying specific and efficient methods to inhibit signal transduction. Previous methods to study this process achieved low success due to low protein yield, poor water solubility of the membrane protein, and the large size of receptor. We report progress on overcoming these technical hurdles by forming the ErbB family members in nanolipoprotein particles (NLPs), which are ∼20 nm disc-shaped cell membrane analogs. Here we compare an optimized cell-based method with a novel cell-free method to produce NLPs containing the ErbB1 receptor, also called EGFR. The cell-produced ErbB1-NLPs retain kinase activity compared to cell-free produced material with higher yields. However, the cell-free produced receptors have regions of correct folding and can be produced in less than a day. The cell-free produced constructs can be labeled conveniently with unnatural amino acids to enable characterization of dimerization interactions. These two receptor production systems can be utilized synergistically to enable new studies of conformational changes associated with ligand binding or protein mutations, as well as to identify small molecule inhibitors of these receptors.

Citation Format: Tiffany M. Scharadin, Christina Takanishi, Wei He, Matthew Saldano, Kermit L. Carraway, Matthew A. Coleman, Paul T. Henderson. Production and characterization of ErbB1 (EGFR) associated with nanolipoprotein particles via cell-based and cell-free methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4275. doi:10.1158/1538-7445.AM2013-4275
Abstract 4277: TRAF2 is a key mediator of the EGF-induced signaling pathway.
Authors: Peng Cong , Zhu Feng , Wen Weihong , Yao Ke , Ma Weiya , Bode Ann , Dong Zigang
Published: August 2014

TRAF2 plays a critical role in the mediation of the TNF-R signaling pathway toward activation of NF-κB and JNKs. Here, we reveal a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage-independent cell transformation. We also showed that TRAF2 is required for RSK2 activity through RSK2 ubiquitination and that RSK2 plays a critical role in AP-1 activity mediated through CREB/c-Fos, which regulate anchorage-independent cell transformation.

Citation Format: Cong Peng, Feng Zhu, Weihong Wen, Ke Yao, Weiya Ma, Ann Bode, Zigang Dong. TRAF2 is a key mediator of the EGF-induced signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4277. doi:10.1158/1538-7445.AM2013-4277
Abstract 4284: Cadherin-11 function is required for full neoplastic transformation of mouse fibroblasts by v-Src.
Authors: Guy Stephanie , Geletu Mulu , Arulanandam Rozanne , Raptis Leda
Published: August 2014

The signal transducer and activator of transcription-3 (Stat3) is an important signal transducer with a role in cancer etiology. Following activation of a number of growth factor or cytokine receptors or expression of oncogenes such as vSrc, Stat3 is phosphorylated at tyr705, dimerizes and migrates to the nucleus where it activates transcription from a number of genes involved in cell division and survival. We recently discovered a novel pathway of activation of Stat3: Engagement of cadherin-11 (cad11), a cell to cell adhesion protein, known to be involved in metastasis, induces a dramatic increase in the levels of the Rac and Cdc42 small GTPases, and this leads to Stat3 activation. Stat3 in turn, downregulates the p53 antioncogene and upregulates anti-apoptotic genes, thus offering a potent survival signal.

A large number of oncogenes such as vSrc activate the E2F transcription factor family, which induce the expression of genes involved in cell division. However, at the same time E2F is a potent apoptosis inducer, hence the high demand of transformed cells for anti-apoptotic signals. Since cad11 activates Stat3, a potent survival factor, we examined the effect of downregulation of cad11 upon transformation of mouse Balb/c3T3 fibroblasts by vSrc. Forced expression of high levels of activated Src caused a dramatic reduction in cad11 levels. However, intermediate Src levels, which are physiologically relevant, did allow cad11 expression, albeit at lower levels. Cad11 knockdown in these cells through shRNA expression with a retroviral vector caused a dramatic reduction in Stat3 activity and induced apoptosis. That is, the residual amounts of cad11 present are sufficient to activate Stat3 and prevent apoptosis induced by the Src/E2F axis. Taken together, these findings indicate that cad11, despite its presumed structural role, and in addition to its known role in promoting metastasis, is actually also necessary for full neoplastic transformation by the Src oncogene.

Citation Format: Stephanie Guy, Mulu Geletu, Rozanne Arulanandam, Leda Raptis. Cadherin-11 function is required for full neoplastic transformation of mouse fibroblasts by v-Src. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4284. doi:10.1158/1538-7445.AM2013-4284
Abstract 4285: Hypoxia-mediated autophagy enhances HGF-induced cancer cell invasion.
Authors: Lee Young H. , Bottaro Donald P.
Published: August 2014

The cellular response to hypoxia is a major influence on the aggressiveness of primary tumors, yet much of how this response mechanism influences other signaling pathways is unknown. The MET proto-oncogene, which encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), is often overexpressed in cancers and can drive cell proliferation, invasion, and metastasis. Prior studies demonstrate that the Met tyrosine-kinase receptor drives cell invasion and metastasis under hypoxic conditions, and this invasive phenotype is lost if MET expression is inhibited. Hypoxia also has been shown to sensitize Met-activated pathways to drive HGF-induced invasion through as yet undefined a molecular mechanism. The high mortality rate of metastatic cancer makes it crucial to understand the functional integration of these two prevalent drivers of metastasis, hypoxia and Met signaling, and to identify ways to selectively block cancer cell invasiveness. We show that a chemically generated pseudo-hypoxic condition enhanced HGF-mediated invasion in the papillary renal cell (PRC) carcinoma cell lines ACHN and UOK112. Under hypoxic conditions, cells generated high levels of ROS, which in turn sensitized the mitogen-activated protein kinase (MAPK) pathway to enhance HGF-driven invasion. Furthermore, hypoxic cells displayed increased autophagy as determined by punctate staining of LC3 II using immunocytochemistry and cleaved LC3 immunoblotting. Inhibition of autophagy decreased cell invasion and hypoxia-mediated autophagy was dependent on MAPK pathway activation. These data suggest that hypoxia promotes the invasiveness of PRC-derived cells by increasing ROS-mediated autophagy to sensitize the MAPK pathway to HGF stimulation. Identifying the specific effectors responsible for the integration these signals will improve our understanding of pro-metastatic invasiveness and potentially identify novels targets for its selective blockade.

Citation Format: Young H. Lee, Donald P. Bottaro. Hypoxia-mediated autophagy enhances HGF-induced cancer cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4285. doi:10.1158/1538-7445.AM2013-4285
Abstract 4294: Copper is required for oncogenic BRAF signaling and tumorigenesis.
Authors: Brady Donita C. , Crowe Matthew S. , Turski Michelle L. , Thiele Dennis J. , Counter Christopher M.
Published: August 2014

The BRAF serine/threonine kinase is mutated, typically at V600, to remain in the active oncogenic state in a large fraction of melanoma, thyroid cancers, and hairy cell leukemia, and to a lesser extent in a wide spectrum of other cancers, thereby activating the kinases MEK1 and MEK2 to stimulate the MAPK pathway and promote cancer. Excitingly, ATP inhibitors of oncogenic BRAF and MEK provide a survival advantage in metastatic melanoma and early clinical studies suggest that coupling BRAF and MEK kinase inhibitors may be even more effective. Thus, the combination of multiple approaches to inhibit MAPK signaling holds great promise for the treatment of BRAF mutation-positive cancers, especially in terms of overcoming resistance. In this regard, we previously found that copper (Cu) influx enhanced MEK1 phosphorylation of its substrates ERK1/2 through a Cu-MEK1 interaction. We show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis, which could be rescued by expressing activated ERK2. Importantly, a Cu chelator used in the treatment of Wilson's disease reduced tumor growth of not only BRAFV600E-transformed cells, but also cells resistant to a BRAF inhibitor. Taken together, these results suggest that Cu-chelation therapy could be repurposed for the treatment of BRAFV600E mutation-positive cancers.

Citation Format: Donita C. Brady, Matthew S. Crowe, Michelle L. Turski, Dennis J. Thiele, Christopher M. Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2013-4294
Abstract 4296: High rates of esthesioneuroblastoma in adenomatous polyposis coli (apc) mutant zebrafish suggest a new modifier mutation altering tissue specificity of Wnt/β-catenin-dependent hyperproliferation.
Authors: Greenwood Vivianne E. , Museles Noah , Weinberg Quinn , Glasgow Eric
Published: August 2014

The most common mutation in human colon cancer is inactivation of adenomatous polyposis coli (APC), a key inhibitor of the Wnt/β-catenin pathway. If left untreated, all humans carrying germline APC-inactivating mutations will develop colon cancer by the age of forty. In addition, truncation of APC is the primary contributor to sporadic colon cancer in humans. In zebrafish, the same truncated mutation in apc also causes digestive tract neoplasia. However, the incidence of tumors outside the intestinal system in the apc mutant zebrafish has not been reported. To determine if other tumors arise at high frequencies in the apc mutant, we monitored several cohorts of zebrafish derived from apc(hu147/+) incrosses on a weekly basis over a one-year period, covering the age of 6 to 18 months post-fertilization. Specimens displaying evidence of tumors or general signs of declining health were euthanized, photographed, tail-clipped for genotypic analysis, and then fixed and embedded in paraffin for histological examination. Analysis of the data showed that 1.2% of the apc(hu147/+) zebrafish had abnormally distended abdomens (possibly from intestinal neoplasia), and 1.8% of the mutants developed olfactory neuroblastoma (esthesioneuroblastoma). We have yet to detect a single wild-type fish with esthesioneuroblastoma in these cohorts. Overall, the results indicate that our apc(hu147/+) zebrafish exhibit a remarkably increased incidence of esthesioneuroblastoma compared to wild-type fish or other tumor-prone mutants. Furthermore, relative to the latter, our apc(hu147/+) zebrafish begin developing tumors of any type at an earlier age. Histopathological examination showed these esthesioneuroblastoma tumors to be highly invasive. No previous reports indicate that ethesioneuroblastoma is a common tumor type in apc(hu147/+) zebrafish. Therefore, we propose to have discovered a new modifier mutation, which leads to hyperproliferation of olfactory neuroepithelium in response to dysregulation of the Wnt/β-catenin pathway. Identification of this mutant gene is expected to shed light on interactions of tissue-specific regulators with components of the Wnt/β-catenin signaling pathway to control rates of cell proliferation and differentiation.

Citation Format: Vivianne E. Greenwood, Noah Museles, Quinn Weinberg, Eric Glasgow. High rates of esthesioneuroblastoma in adenomatous polyposis coli (apc) mutant zebrafish suggest a new modifier mutation altering tissue specificity of Wnt/β-catenin-dependent hyperproliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4296. doi:10.1158/1538-7445.AM2013-4296
Abstract 430: Inhibition of Pin1 reduces RAD51 expression and enhances radiosensitivity in U87MG glioblastoma cells.
Authors: Chen Edmund I. I. , Ho Chia-Hsin
Published: August 2014

Glioblastoma multiforme (GBM) is the most common primary brain tumor, and has poor prognosis and high recurrences. Pin1, a peptidyl-prolyl isomerase, can recognize phospho-Ser/Thr-Pro motifs and isomerize the cis/trans forms of its substrates. It has been known that Pin1 is involved in cell cycle regulation and cell proliferation, and has affected various protein stability and function, such as cyclin D1 and p53. Recent study has showed that cyclin D1 can directly bind to RAD51 upon radiation in addition to its role in cell cycle. This complex is recruited to DNA damage sites and helps DNA repair. Here, we investigated the role of Pin1 in regulating cyclin D1/RAD51 interaction and affecting the radiosensitivity in U87MG glioblastoma cells. We showed that no interaction was detected between cyclin D1 and RAD51 after ionic radiation in U87MG cells; interestingly, knockdown of Pin1 decreased both cyclin D1 and RAD51 expression and enhanced their sensitivities to radiation. Furthermore, knockdown of Pin1 with IR induced G1 and G2/M arrest which resulted in cell growth inhibition and apoptosis. Importantly, knockdown of Pin1 and combination with radiation yielded increased number of γ -H2AX foci than radiation control cells, suggesting that delayed repair occurred in pin1 knockdown cells. In summary, our results suggest that inhibition of Pin1 decreases RAD51 expression and enhances radiosenstivity in U87MG glioblastoma Cells.

Citation Format: Edmund I. IT Chen, Chia-Hsin Ho. Inhibition of Pin1 reduces RAD51 expression and enhances radiosensitivity in U87MG glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 430. doi:10.1158/1538-7445.AM2013-430
Abstract 4302: TACC3 promotes EMT through the up-regulation of PI3K/Akt and ERK signaling pathways.
Authors: Ha Geun-Hyoung , Park Jong-Sup , Breuer Eun-Kyoung Y.
Published: August 2014

Transforming acidic coiled-coil protein 3 (TACC3) is a member of TACC family, essential for the maintenance of centrosome integrity and the regulation of microtubule assembly and spindle stability during mitosis. In addition to its function in mitosis, TACC3 has been shown to be involved in controlling cell growth, differentiation and transcriptional regulation. Interestingly, deregulated expression of TACC3 has been found in many human cancers, including breast, ovary, bladder, glioblastoma and non-small cell lung cancer. Moreover, our previous microarray analysis suggested that overexpression of TACC3 may be associated with the progression, metastasis and chemoresistance of cervical cancer. Although these findings indicate that TACC3 may play an important role in human cancer, the fundamental molecular mechanisms underlying how TACC3 contributes to cancer initiation, progression and metastasis, and its clinical significance remain elusive. Here, we explored the significance of TACC3 in cervical cancer. Using cervical cancer tissue microarrays, we found that TACC3 is overexpressed in cervical cancer compared with normal cervix. By modulating the expression of TACC3, we found that overexpression of TACC3 leads to changes in cell morphology, proliferation, transforming capability, migratory/invasive behavior as well as the expression of epithelial-mesenchymal transition (EMT)-related markers. Moreover, phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinases (ERKs) signaling pathways are critical for TACC3-mediated EMT process. Notably, depletion of TACC3 is sufficient to suppress EMT phenotype. Our findings, therefore, identify a novel mechanism that promotes EMT, possibly leading to a more malignant and metastatic tumor phenotype as well as a potential biomarker for diagnosis, prognosis and therapy for cervical cancer.

Citation Format: Geun-Hyoung Ha, Jong-Sup Park, Eun-Kyoung Y. Breuer. TACC3 promotes EMT through the up-regulation of PI3K/Akt and ERK signaling pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4302. doi:10.1158/1538-7445.AM2013-4302
Abstract 4313: A LIM protein ajuba suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade.
Authors: Tanaka Ichidai , Osada Hirotaka , Fujii Makiko , Sekido Yoshitaka
Published: August 2014

Malignant mesothelioma (MM) is a highly aggressive neoplasm which is associated with asbestos exposure and around 50% of MM tumors have a mutation of the NF2 tumor suppressor gene. NF2 encodes merlin, a member of the ezrin/radixin/moesin protein family, and merlin is an upstream regulator of the Hippo signaling cascade. Activation of the Hippo pathway induces the activation of serine/threonine kinases LATS1/2, which phosphorylate and inactivate a transcriptional coactivator, YAP. The Hippo pathway activation negatively regulates tissue growth and its inactivation is involved in carcinogenesis.

We previously found that, besides NF2 mutation, a subset of MM tumors harbor LATS2 mutation or YAP oncogene amplification. However, there are yet other MM cases that don't have any of these gene mutations, leading us to hypothesize that other components of this pathway might be altered. Recently, the LIM protein Jub in Droshophila was shown to activate Yoki (ortholog of human YAP) through suppression of the Hippo pathway. To determine whether the human ortholog of Jub, Ajuba, is involved in the Hippo signaling downregulation in MM, we analyzed 20 MM cell lines for the alteration of Ajuba.

We found that the expression of Ajuba was significantly downregulated in 6 of 20 MM cell lines compared to an immortalized normal mesothelial cell line, Met-5A. Interestingly, three of the 6 cell lines with low Ajuba expression showed YAP activation, whereas Met-5A cells showed high Ajuba expression and YAP inactivation, both of which suggested that Ajuba might act as a tumor suppressor instead of oncogenic protein in mesothelial cells. We infected Ajuba-expressing lentivirus into these MM cell lines and found that exogenous Ajuba induced YAP phosphorylation and inhibited MM cell proliferation. Since the CCND1 and connective tissue growth factor (CTGF) genes are well-known transcriptional targets of YAP, we conducted a dual-luciferase reporter assay using these promoter regions to see if Ajuba affects their transcriptional activities. The dual-luciferase assay demonstrated that Ajuba suppressed the promoter activity of these genes and that the suppression of them was attenuated by LATS2 knockdown. These results indicated that Ajuba negatively regulates YAP through activation of LATS2. Our results suggest that the inactivation of Ajuba is one of the mechanisms for constitutive activation of YAP, which results in deregulation of MM cell proliferation.

Citation Format: Ichidai Tanaka, Hirotaka Osada, Makiko Fujii, Yoshitaka Sekido. A LIM protein ajuba suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2013-4313
Abstract 4314: Loss of CYLD promotes cell migration through stabilized ALK5 in human oral squamous cell carcinoma cells.
Authors: Shinriki Satoru , Jono Hirofumi , Li Jian-Dong , Nakamura Takuya , Hayashi Mitsuhiro , Guo Jianying , Shinohara Masanori , Ando Yukio
Published: August 2014

Five-year survival of patients with oral squamous cell carcinoma (OSCC) has not changed apparently for the past 30 years. Although cylindromatosis (CYLD) is thought as a potent tumor suppressor, its biological significances in malignancies are largely unknown. The aim of this study was to clarify the role of CYLD in OSCC progression. We investigated expression of CYLD in OSCC including intraepithelial neoplasia (IEN; n = 48) and invasive carcinomas (n = 133) tissues and normal oral mucosal tissues (n = 35) by immunohistochemistry. In addition, effects of CYLD knockdown by siRNA or its overexpression on OSCC progression using OSCC cell lines. Our immunohistochemical analyses revealed that CYLD expression was significantly reduced at invasive lesions in OSCC tissues whereas it was conserved in normal epithelium and IEN. In addition, lower CYLD expression was associated with poor overall survival in invasive OSCC. Accordingly, CYLD knockdown led to acquisition of mesenchymal state and increased migratory activity in OSCC cells. Interestingly, loss of CYLD enhanced TGFβR1 (ALK5) protein expression by promoting ALK5 stability while overexpression of wild-type CYLD downregulated ALK5. Furthermore, EMT-like changes induced by CYLD knockdown were completely blocked by a TGFβR1 kinase inhibitor in OSCC cells. These findings suggest that downregulation of CYLD promotes invasion through EMT-like changes via ALK5 stabilization in OSCC cells. Further studies for these mechanisms probably provide new insights into the TGFβ signal transduction in cancer and development of a novel therapy in OSCC.

Citation Format: Satoru Shinriki, Hirofumi Jono, Jian-Dong Li, Takuya Nakamura, Mitsuhiro Hayashi, Jianying Guo, Masanori Shinohara, Yukio Ando. Loss of CYLD promotes cell migration through stabilized ALK5 in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2013-4314
Abstract 4317: PTEN-null tumors cohabiting the same lung display differential Akt activation and sensitivity to dietary restriction.
Authors: Curry Natasha L. , Mino-Kenudson Mari , Oliver Trudy G. , Yilmaz Omer H. , Yilmaz Vedat O. , Moon Jade , Jacks Tyler , Sabatini David M. , Kalaany Nada Y.
Published: August 2014

PTEN loss is considered a biomarker for activated PI3K/Akt, a pathway frequently mutated in cancer, and recently shown to confer resistance to dietary restriction (DR). Here we demonstrate that PTEN loss is not sufficient to drive Akt activation and resistance to DR in tumors with low growth factor receptor levels. We describe a murine PTEN-null Kras-driven lung cancer model that harbors both DR-resistant, higher-grade, bronchiolar tumors with high-Akt-activity, and DR-sensitive, lower-grade, alveolar tumors with low-Akt-activity. We find that this phenotype is cell-autonomous and that normal bronchiolar cells express higher levels of IGF1R and ENTPD5, an endoplasmic reticulum (ER) enzyme known to modulate growth factor receptor levels. Suppression of ENTPD5 is sufficient to decrease IGF1R levels and sensitize bronchiolar tumor cells to serum in vitro and to DR in vivo. Furthermore, we find that a significant percentage of human non-small cell lung cancer (NSCLC) have low Akt activity despite PTEN loss. Our studies point to a heterogeneity of Akt activation in the same murine PTEN-null lung tissue, as well as in human NSCLC, underscoring the challenges facing personalized cancer therapy based solely on cancer genotype. They further indicate that the tumor response to anti-cancer therapies, including DR, needs to be based upon PI3K/Akt activity per se, rather than genetic alterations in the PTEN/Akt pathway.

Citation Format: Natasha L. Curry, Mari Mino-Kenudson, Trudy G. Oliver, Omer H. Yilmaz, Vedat O. Yilmaz, Jade Moon, Tyler Jacks, David M. Sabatini, Nada Y. Kalaany. PTEN-null tumors cohabiting the same lung display differential Akt activation and sensitivity to dietary restriction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4317. doi:10.1158/1538-7445.AM2013-4317
Abstract 4325: Monoclonal antibodies to transmembrane proteins.
Authors: Brown Michael C. , Chambers Ross , Onisk Dale V. , Joaquim Tony R. , Stafford Lewis J. , Lindpaintner Klaus , Keter Daniel , Stave James W.
Published: August 2014

Transmembrane proteins, including multipass transmembrane proteins like GPCRs and ion channels, are important targets for therapeutic monoclonal antibody (mab) discovery. Therapeutic antibodies to this class of proteins are generally targeted to extracellular domains displayed on the surfaces of living cells. Challenges associated with developing antibodies to this class of targets are small numbers of extracellular amino acids, membrane-dependent protein conformation, difficulty in expression at high levels, high amino acid sequence homology of human and mouse proteins, and post-translational modifications. DNA immunization strategies with full-length constructs and high throughput flow cytometry screening of mab binding to transfected and control cells was used to generate and identify large numbers of mabs to CXCR4 and ADORA2A (GPCRs) and CD20 (a tetraspan membrane protein). Panels of mabs were generated for all 3 targets with low numbers of hybridoma fusions. For each target the mab gene sequences were shown to be unique and contain levels of somatic hypermutation comparable to existing benchmark therapeutic antibodies. Epitope mapping with mutant proteins identified diverse patterns of reactivity including known and novel specificities. Functional assays including apoptosis and receptor modulation (calcium flux and cAMP modulation) further demonstrated that the technical approach generated diverse panels of antibodies that exhibit functional activity as good or better than existing benchmark therapeutic antibodies

Citation Format: Michael C. Brown, Ross Chambers, Dale V. Onisk, Tony R. Joaquim, Lewis J. Stafford, Klaus Lindpaintner, Daniel Keter, James W. Stave. Monoclonal antibodies to transmembrane proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2013-4325
Abstract 4331: Advantages of polyacetal polymer-based antibody drug conjugates employing cysteine bioconjugation.
Authors: Yurkovetskiy Alex , Bodyak Natalya , Yin Mao , Thomas Joshua , Conlon Patrick , Stevenson Cheri , Uttard Alex , Qin Liu , Gumerov Dmitry , Ter-Ovaneysan Elena , DeVit Michael , Lowinger Timothy B.
Published: August 2014

The application of polymers to antibody drug conjugate (ADC) design and preparation can provide numerous advantages, including 1) significantly higher capacity for drug payload; 2) utilization of alternative payloads not suitable for direct conjugation approaches; 3) improvement of physicochemical properties of resulting ADCs; 4) utilization of protein recognition scaffolds beyond the commonly used IgGs; and 5) improvements in PK and biodistribution. Herein we present results of a novel, biodegradable polyacetal polymer-based conjugation system to create next-generation ADCs. The basis of this new conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal) modified with chemically orthogonal linkers. One linker is used to covalently attach a targeting moiety (mAb or alternative) via cysteine conjugation, while a second, chemically distinct linker is used to attach a drug payload and to control the mechanism and rate of drug release.

Previously we have reported highly efficacious polyacetal ADCs prepared by utilizing random lysine modification. In this report we present an alternative cysteine-based bioconjugation strategy. Conventional, direct drug-cysteine linked ADCs are destabilized due to loss of inter-chain disulfide bridges. In contrast, the polyacetal polymer conjugated via cysteines in the antibody hinge region stabilizes the resulting ADCs through the formation of inter-chain bridge structures.

To demonstrate the benefits of this approach, we prepared Her-2 targeted ADCs with protein recognition scaffolds ranging in size from 15 kDa to 150 kDa, all targeting the Her-2 antigen, and bearing an anti-tubulin agent as the drug payload. ADCs were highly active and selective in vitro in Her-2 expressing cell lines. These polyacetal cysteine-based ADCs exhibited prolonged plasma and tumor exposure in the Her-2 expressing BT474 mouse xenograft model and tumor-specific accumulation. The ADCs were well-tolerated, and resulted in 100% tumor-free survivors.

Citation Format: Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Joshua Thomas, Patrick Conlon, Cheri Stevenson, Alex Uttard, Liu Qin, Dmitry Gumerov, Elena Ter-Ovaneysan, Michael DeVit, Timothy B. Lowinger. Advantages of polyacetal polymer-based antibody drug conjugates employing cysteine bioconjugation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4331. doi:10.1158/1538-7445.AM2013-4331
Abstract 4333: Self-stabilizing ADCs: antibody-drug conjugates prepared with maleimido drug-linkers that catalyze their own thiosuccinimide ring hydrolysis.
Authors: Lyon Robert P. , Setter Jocelyn R. , Bovee Tim D. , Doronina Svetlana O. , Anderson Martha E. , Leiske Chris L. , Senter Peter D.
Published: August 2014

Many antibody-drug conjugates (ADCs) currently in clinical trials employ maleimide-containing drug-linkers which are conjugated to antibody cysteine residues to form thiosuccinimide linkages. It is now known that these thiosuccinimide linkages can undergo two competing reactions while in plasma: elimination of the maleimide resulting in undesirable loss of drug from the ADC, and hydrolysis of the thiosuccinimide ring resulting in a succinic acid derivative which cannot undergo elimination. Thus, thiosuccinimide ring hydrolysis is a reaction which stabilizes the chemical linkage of the drug to the antibody. We have engineered a new class of drug-linkers which incorporate a basic amino group adjacent to the maleimide, providing intramolecular base catalysis of the ring hydrolysis. This basic group induces the thiosuccinimide to undergo rapid self-catalyzed hydrolysis at neutral pH and room temperature, with complete hydrolysis achieved in less than 2 hours. Once hydrolyzed, the drug-linker is no longer subject to maleimide elimination reactions, preventing loss of drug from the antibody by this mechanism and forming a highly stable ADC. To compare these new drug-linkers to traditional maleimido drug-linkers, a number of in vitro and in vivo studies have been conducted. Results of these studies will be presented to demonstrate a robust improvement in drug-linker stability compared to traditional drug-linker formats.

Citation Format: Robert P. Lyon, Jocelyn R. Setter, Tim D. Bovee, Svetlana O. Doronina, Martha E. Anderson, Chris L. Leiske, Peter D. Senter. Self-stabilizing ADCs: antibody-drug conjugates prepared with maleimido drug-linkers that catalyze their own thiosuccinimide ring hydrolysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4333. doi:10.1158/1538-7445.AM2013-4333
Abstract 4334: Identification and elimination of T-cell epitopes in recombinant immunotoxins used to treat cancer.
Authors: Mazor Ronit , Vassall Aaron N. , Eberle Jaime A. , Beers Richard , Hu Xiaobo , Benhar Itai , Pastan Ira
Published: August 2014

Moxetumomab pasudotox and SS1P are immunotoxins that have been developed in our lab to treat cancer. Moxetumomab pasudotox, which can be given for many treatment cycles, targets CD22 and has produced many complete remissions in drug-resistant hairy cell leukemia. However, SS1P, which targets mesothelin, could only be given for one cycle before ADA (anti drug antibodies) developed and neutralized the protein. We hypothesize that SS1P could be more effective if it were less immunogenic and more treatment cycles could be given.

To de-immunize the immunotoxin we sought to identify and remove T cell epitopes. To identify the epitopes we stimulated PBMCs from 50 normal donors with whole immunotoxin for 14 days to expand the T cells and then stimulated the T cells with peptides (15 mers) derived from the toxin, followed by ELISpot to identify the peptides that stimulated the T cells. We found that all 50 donors gave a positive response, which correlates with the frequency of ADAs in patients with normal immune systems. We used alanine-scanning mutagenesis to determine which amino acids in each peptide are important for T cell stimulation.

SS1P contains domains II and III of PE. In 46% of the donors we found a single highly immunodominant and promiscuous epitope that is located in domain II. We previously showed we could eliminate almost all of domain II while retaining excellent cytotoxic activity. These data indicate that immunogenicity should be eliminated in 34% of normal individuals donors and diminished in another 42% by deletion of domain II. In domain III we identified 5 peptides that stimulated T cell responses in different donors and also identified alanine mutations within each peptide that eliminated or greatly lowered T cell stimulation in most donors. We are using this information to make immunotoxins with mutations in domain III that should have a further decrease in immunogenicity.

We have shown that among many individuals with different HLA alleles there is a dominant T cell epitope in domain II of PE38 and 5 subdominant epitopes in domain III. We plan to use this information to make new immunotoxins in which the large majority of T cell epitopes are silenced resulting in diminished immunogenicity of the immunotoxin. We plan to combine these T cell mutations with B cell mutations previously described to make immunotoxins with very low immunogenicity allowing more treatment cycles to be given and better anti-tumor activity achieved.

Citation Format: Ronit Mazor, Aaron N. Vassall, Jaime A. Eberle, Richard Beers, Xiaobo Hu, Itai Benhar, Ira Pastan. Identification and elimination of T-cell epitopes in recombinant immunotoxins used to treat cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2013-4334