bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract PL06-04: Molecular characterization of circulating tumor cells.
Authors: Haber Daniel A. , Maheswaran Shyamala , Sequist Lecia , Yu Min , Ozkumur Emre , Ting David , Miyamoto David , Lee Richard , Shah Ajay , Bardia Aditya , Stott Shannon , Toner Mehmet
Published: August 2014

Circulating tumor cells (CTCs) are present at extraordinarily low levels in the blood of patients with non-hematological cancers, but they provide a non-invasive source of cancer cells for potential diagnostic applications, as well as for understanding the process of blood-borne metastasis. We have developed and tested three generations of microfluidic devices for the isolation of CTCs, each with further improvements in automation, processing throughput, and downstream analytical capabilities. The CTC-iChip allows high throughput and high efficiency depletion of normal leukocytes through a microfluidic magnetophoresis device, revealing untagged CTCs. The unbiased isolation of CTCs irrespective of tumor epitopes, together with their presentation in a format where they can be readily manipulated, provides unique opportunities for their detailed molecular characterization. We have previously shown that rising or declining numbers of CTCs are well correlated with tumor progression or therapeutic response; that they can be used to monitor the prevalence of known clinically-relevant mutations; and that changes in their composition can be followed longitudinally during the course of targeted cancer therapies. Epithelial-to-mesenchymal transition (EMT) is evident among CTCs and evolves dynamically during the course of response or progression following cancer therapy. The heterogeneity of CTC populations is evident in single cell imaging analyses and sequencing studies, with implications for understanding the complexity of metastatic progression and therapeutic responses. Taken together, the application of novel bioengineering technologies to the isolation and analysis of CTCs across multiple different cancer types offers a unique window into the properties of cancer cells that invade into the bloodstream and give rise to metastases.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL06-04.

Citation Format: Daniel A. Haber, Shyamala Maheswaran, Lecia Sequist, Min Yu, Emre Ozkumur, David Ting, David Miyamoto, Richard Lee, Ajay Shah, Aditya Bardia, Shannon Stott, Mehmet Toner. Molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL06-04.
Abstract PL07-01: Immune checkpoint blockade in cancer therapy: New insights and opportunities.
Authors: Allison James P.
Published: August 2014

The existence of multiple non-redundant l inhibitory pathways that limit T cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen presenting cells. Antibodies to CTLA-4 have proven effective against multiple tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long term (>4.5 years) survival benefit in about 23% of patients in a randomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by the FDA for treatment of late stage melanoma and is now a standard of care for that disease. The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However, there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) and regulatory (Treg) T cells. We have recently uncovered a mechanism whereby anti-CTLA-4 antibodies expand Treg in lymph nodes but cause their depletion in the tumor microenvironment. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple mechanisms. PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25%. A recent Phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late stage melanoma patients. These studies and their implications for cancer therapy will be discussed.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL07-01.

Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights and opportunities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL07-01.
Abstract 1854: Effects of supplemental antioxidant micronutrients on oxidative DNA damage marker in normal colorectal mucosa: A randomized clinical trial
Authors: Lakkur Sindhu , Ahearn Thomas , Bostick Roberd
Published: August 2014

Background: Basic science and animal studies suggests that oxidative stress plays a role in colorectal oncogenesis. Smokers are reported to have increased oxidative DNA lesions in transformed colorectal epithelial cells and increased colorectal cancer risk; however, the majority of observational and clinical studies report weak or null associations between oxidative stress and risk of colorectal neoplasia. To address this we conducted, a pilot, randomized, double blinded, placebo controlled clinical trial to test the effects of an antioxidant micronutrient combination on a marker of oxidative DNA damage, 8-hydroxy-2’-deoxyguanosine (8-OH-dG), in the normal colorectal mucosa.

Methods: Forty-seven patients diagnosed with at least one pathology confirmed sporadic colorectal adenoma were given a daily antioxidant micronutrient combination (800mg dl-alpha-tocopherol acetate, 24 mg beta carotene, 1.0 g vitamin C, 200µg l-selenomethionine, 7.2 mg riboflavin, 80 mg niacin, 60 mg zinc, 5 mg manganese) or placebo for 4 months. A subset of patients (n=33) for whom tissue was available pre- and post supplementation was used in this analysis. 8-OH-dG was detected in biopsies of normal-appearing colorectal mucosa using standardized automated immunohistochemistry. Overall labeling and distribution of 8-OH-dG within the colon crypts were quantified by novel image analysis methods. Treatment effects were assessed using a MIXED effects model as implemented in SAS v9.2.

Results: Relative to the placebo group, in the antioxidant group 8-OH-dG labeling decreased 13% (p=0.87) along the full length of crypts, 27% (p=0.49) in the upper third of crypts, 27 % (p=0.53) in the middle third of crypts, and 8% (p=0.92) in the bottom third of crypts. Among smokers (n=14), relative to the placebo group, in the antioxidant group 8-OH-dG labeling increased 6% (p=0.82) in the upper third of crypts, 28% (p=0.65) in the middle third of crypts, and 51% (p=0.50) in the bottom third of crypts. In non-smokers (n=18), relative to the placebo group, in the antioxidant group 8-OH-dG expression decreased 23% (p=0.55) in the upper third of crypts, 34% (p=0.44) in the middle third of crypts, and 24% (p=0.57) in the bottom third of crypts.

Discussion: These preliminary findings, while not statistically significant 1) suggest that antioxidants may modulate oxidative DNA damage in the normal-appearing human colorectal epithelium – potentially modified by smoking status; 2) provide support for the use of 8-OH-dG as a potential treatable biomarker of risk for colorectal neoplasms; and 3) provide support for extending this investigation in a larger, more definitive trial.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1854. doi:10.1158/1538-7445.AM2011-1854
Abstract 1934: Molecular mechanism of abnormal localization of nucelolin on cell-surface of gastric cancer cells, with regard to Ras signaling pathway
Authors: Watanabe Tatsuro , Hirano Kazuya , Beppu Masatoshi , Yamaguchi Kensei , Takahashi Atsushi , Fujiki Hirota , Suganuma Masami
Published: August 2014

Our intensive study of TNF-α inducing protein (Tipα), a carcinogenic factor secreted from H. pylori, revealed that nucleolin acts as a receptor of Tipα in a mouse gastric cancer cell line (MGT-40). Although nucleolin is a major nucleolar protein, nucleolin was found in the cell membrane by cell fractionation and on the cell-surface by flow cytometrical analysis. We further found that nucleolin shuttles Tipα from cell surface to the cytosol, resulting in expression of TNF-α gene, an endogenous tumor promoter. Moreover, a large amount of nucleolin was found in the membrane fraction of MGT-40 cells, but not in that of mouse normal glandular stomach. These results suggest that abnormal localization of nucleolin occures in gastric cancer, as an associated process of carcinogenesis. In the light of this evidence, we think that the abnormal localization of nucleolin on cell surface produces the optimal carcinogenic environment for Tipα binding in gastric cancer development by H. pylori infection. Experimentally we found that large amounts of nucleolin were expressed on the surface of four human gastric cancer cell lines (MKN-45, MKN-74, AGS, and KATOIII), with smaller amounts on that of another cell line (MKN-1), although the total amounts of nucleolin were almost the same among these five cell lines. It is important to note that the amounts of cell-surface nucleolin are correlated well with those of Ras-GTP form, the active form of Ras protein in these cell lines, and we confirmed that MKN-1 cells had the lowest amounts of Ras-GTP form among the five lines. Moreover, the treatment of AGS cells with okadaic acid, a tumor promoter and inhibitor of protein phosphatases 1 and 2A, reduced the amount of nucleolin localized on cell-surface, indicating that abnormal localization of nucleolin on cell surface can be induced by Ras signaling pathway and post-translational modifications. Thus, we discuss the relationship between the abnormal localization of nucleolin and the disorder in the Ras signaling pathway in gastric cancer development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2011-1934
Abstract 196: Functional role of death-associated protein 3 in high grade gliomas of childhood
Authors: Vladimirova Valentina A. , Soerensen Niels , Pietsch Torsten
Published: August 2014

Background: High grade gliomas (HGG) are highly invasive tumors and respond poorly to conventional treatments. Although significant progress has been made in understanding the molecular pathways that lead to the development of HGG in adults, comparatively few data is available for gliomas of childhood. The death-associated protein 3 (DAP3) is localized on the chromosomal region 1q21-22 that has been previously described as a region of frequent chromosomal gain associated with poor outcome in HGG. Moreover, DAP3 has been reported to be overexpressed and to play anti-apoptotic role in invasive glioblastoma cells.

Methods: In the current study to investigate the functional role of DAP3 in pediatric versus adult HGG, we suppressed the endogenous DAP3 protein using DAP3-specific short hairpin RNA (shRNA) or chemical inhibitors of protein kinase C, and analyzed the phenotypic effect on glioma cell proliferation, migration, mitochondrial morphology and apoptosis.

Results: We found that knockdown of DAP3 by shRNA induced mitochondrial fragmentation and apoptosis in pediatric glioma cell lines while in adult glioma cell lines it suppressed cell proliferation and reduced mitochondrial potential without inducing mitochondrial fragmentation. Specific chemical inhibition of the two classical PKC isoforms (PKCα and PKCβ) significantly reduced the expression of DAP3 protein in both pediatric and adult glioma cell lines. The inhibition of PKCα changed cell morphology, mitochondrial status and significantly suppressed proliferation and migration of both pediatric and adult glioma cell lines without associated caspase activation indicating that DAP3 may mediate nonapoptotic form of cell death in HGG. The suppression of PKCβ rendered pediatric glioma cells more sensitive to the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) through a caspase-dependent process as compared with adult glioma cells suggesting a possible implication of DAP3 in intrinsic and extrinsic pathways for apoptosis in pediatric HGG.

Conclusion: Our findings confirmed the anti-apoptotic role of DAP3 in glioma cell biology suggesting that specific PKC isoforms mediated the DAP3 suppression of apoptosis in HGG and pointed out the specific functional role of this protein in mitochondrial maintenance. DAP3 may represent a novel target for the treatment of gliomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 196. doi:10.1158/1538-7445.AM2011-196
Abstract 1960: Phosphorylation of caspase-7 by p21-activated protein kinase (PAK)2 inhibits staurosporine-induced apoptosis of MCF-7 breast cancer cells
Authors: Zhu Feng , Li Xiang , Wen Weihong , Liu Kangdong , Malakhova Margarita , Peng Cong , Kim Hong-Gyum , Ma Weiya , Cho Yeon-Yong , Bode Ann , Dong Ziming , Dong Zigang
Published: August 2014

P21-activated kinase (PAK) 2, a member of PAK family of serine/threonine kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in chemotherapy resistance, as well as cell proliferation, is not clear. In this report, we showed that the protein expression level of PAK2 is higher in human breast cancer cell lines and human breast invasive carcinoma tissues compared with adjacent normal breast tissues. Importantly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at Ser30, Thr173 and Ser239. Phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Knocking down PAK2 expression can restore caspase-7 activity and promote apoptosis of MCF-7 breast cancer cells. Our data suggests that the highly expressed PAK2 mediates chemotherapy resistance in human breast invasive ductal carcinoma and that PAK2 is a novel target of chemotherapy for breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2011-1960
Abstract 1966: Regulation of RKIP function by Helicobacter pylori in gastric cancer cells
Authors: Moen Erika , Anwar Talha , Wen Sicheng , Onikoyi Omobola , Moss Steven , Chatterjee Devasis
Published: August 2014

Raf kinase inhibitory protein (RKIP) is a negative regulator of the mitogen activated protein kinase (MAPK) cascade initiated by Raf-1 and nuclear factor kappa B (NF-κB) and is an activator of apoptosis in human cancers. STAT3 (signal transducer and activators of transcription) is often constitutively phosphorylated in gastric cancer. RKIP is a negative regulator of STAT3 and has been positively correlated with a higher survival rate in gastric cancer patients (Chatterjee, et al., Clin. Cancer Res., 14:2994-3001, 2008). Helicobacter pylori (H. pylori) infection is associated with high levels of activated STAT3 as well as activation of the transcription factor NF-κB in gastric epithelial cells, through mechanisms involving macrophage-derived IL-6. H. pylori infection led to a dose- and time-dependent increase in STAT3 activation and Snail, a protein that is a negative regulator of RKIP transcription. In addition, H. pylori and IL-6 treatment stimulated the phosphorylation of RKIP in a protein kinase C- (PKC) dependent manner. Interestingly, H. pylori stimulated RKIP transcription which was enhanced in the presence of the histone acetyl transferase, CBP, and inhibited in the presence of the PKC inhibitor. Surprisingly, RKIP stimulated STAT3 transcription in gastric cancer cells, an effect that was reversed in the presence of CBP. After H. pylori infection, RKIP localized to the nucleus and enhanced H. pylori-mediated apoptosis. Collectively, these results indicate that RKIP function is regulated by phosphorylation after H. pylori infection and acetylation by CBP. Moreover, our study demonstrates that RKIP post translational modifications regulate apoptosis induction (via phosphorylation) and inhibition of cell survival (via acetylation) in gastric cancer. These results also suggest the existence of a feedback loop between RKIP, H. pylori infection and STAT3 in gastric cancer. Supported, in part, by NIH COBRE Investigator Award Grant P20 RR17695 (DC and SM).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1966. doi:10.1158/1538-7445.AM2011-1966
Abstract 197: Multi-cellular tumor spheroids in vitro model reveals a critical role of ERBB4 in survival of osteosarcoma
Authors: Hua Yingqi , Yang Yanwen , Hughes Dennis
Published: August 2014

Multi-cellular tumor spheroids (MCTS) model is an in vitro model holding an intermediate complexity between monolayer culture and solid tumor. It is cell-cell adhesion dependent and closely resembles avascular tumor regions and micrometastasis in pro-angiogenesis stage. Osteosarcoma (OS) is the most common malignant bone tumor with high incidence of early lung metastasis; therefore, multi-cellular tumor spheroid can be used to model its in vivo behaviors. Our group previously reported the expression profile of ERBB family in osteosarcoma tissue samples and cell lines. To further study ERBB pathways in osteosarcoma tumor spheroids, we generated OS tumor spheroids by plating cells on non-adherent poly-HEMA pre-coated dishes. Under anchorage-independent conditions, OS cells spontaneously generate multi-cellular spheroids to acquired anoikis resistant by cell-cell adhesion; blocking the formation of spheroids will result rapid cell death and increased PARP cleavage. MCTSs showed lower proliferation rate than monolayer culture, and they held a chemo-resistant phenotype, as shown by proliferation assay and subdiploid DNA content. Surprisingly, ERBB4 expression was up-regulated in non-adherent tumor spheroids compared with monolayer culture (1.2, 2.9, 6.3 fold respectively in 3 cell lines) while EGFR and ERBB2 did not. With knockdown of ERBB4 expression by shRNA, OS cells became less tolerant to anoikis and serum starvation, as shown by PARP cleavage and subdiploid DNA content. Moreover, knockdown of ERBB4 increased the chemo-sensitivity of OS cells against multiple chemotherapy including cisplatin, doxorubicin, and methotrexate (71%, 71%, 60% in survival compared to scramble control). In conclusion, our data showed that ERBB4 contributes to the tumorigenicity of osteosarcoma and plays a protective role against multiple exogenous apoptotic stimuli including matrix detachment, nutrition insufficiency and cytotoxic reagents. Although wildly expressed in osteosarcoma, the role of ERBB4 in tumor biology is largely unknown. These findings indicate that ERBB4 is a key factor regulating survival pathway of OS under various cellular stress condition. Thus, in primary osteosarcoma and metastatic lesion, ERBB4 might be a potential therapeutic target to facilitate multi-modal therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 197. doi:10.1158/1538-7445.AM2011-197
Abstract 1975: Discovery of a potent and selective Jak2 inhibitor that suppresses GM-CSF-induced STAT5 phosphorylation and Erythropoietin-induced reticulocytosis in vivo
Authors: Liu Liqin , Moody Gordon , Pistillo Jeanne , Emkey Renee , Yu Violeta , Doherty Elizabeth M. , Huang Xin , Kim Joseph L. , Sinclair Angus M.
Published: August 2014

Essential thrombocythemia (ET), polythemia vera (PV) and myelofibrosis (MF) are myeloproliferative disorders (MPDs) characterized by a chronic excessive production of cells from one or more myeloid lineages and/or bone marrow fibrosis, and have the potential to progress to AML. Recently, gain-of-function mutations in intracellular tyrosine kinase Janus kinase 2 (Jak2) were identified to be associated with MPDs. Jak2 is a member of the Jak family of kinases including Jak1, Jak3, and Tyk2 and is the most proximal signaling component for a number of cytokine receptors. The most common Jak2 mutation identified in MPDs is a substitution of valine to phenylalanine at codon 617 (V617F) located in the pseudokinase domain, which results in the loss of its repressive function, and subsequently leads to the constitutive activation of Jak2 and downstream signaling pathways (STAT, MAP kinase, and PI3 kinase) which affect the survival, differentiation and proliferation of hematopoietic progenitors.

Highly selective Jak2 inhibitors may provide a better therapeutic window in chronic dosing settings (ET and PV patients) than non-selective Jak inhibitors. Here we report the discovery of a potent and highly selective Jak2 inhibitor, AMG-Jak2-01. In enzyme assays, AMG-Jak2-01 was potent (3 nM) and selective over other Jak family kinases (> 2000-fold over Jak1, 10-fold over Jak3 and > 400-fold over Tyk2). In isogenic BaF3 cell lines expressing constitutively active Jak kinases (Tel-Jak fusion), AMG-Jak2-01 showed over 40-fold Jak2 selectivity against Jak1 or Tyk2 and 10-fold selectivity against Jak3 based on the inhibition of STAT5 phosphorylation (pSTAT5 – detected with AlphaScreen®). In cytokine-stimulated primary PBMC or whole blood assays, AMG-Jak2-01 demonstrated more than 25-fold Jak2 selectivity over Jak1and Jak3 based on inhibition of pSTAT5 (detected by flow cytometry). In vivo, oral administration of AMG-Jak2-01 at 100 mg/kg for 1 hr inhibited GM-CSF-induced pSTAT5 by approximately 70% (p<0.01) in a mouse peripheral blood pharmacodynamic assay. Finally, two doses of AMG-Jak2-01(100 mg/kg) given 4 hrs apart were able to suppress Epo-induced reticulocytosis in the mouse by 70% (p<0.02).

In summary, we have discovered a potent Jak2 inhibitor, AMG-Jak2-01, which is selective over other Jak family members in both enzyme and cell-based assays and demonstrates inhibitory activity in GM-CSF-induced STAT5 phosphorylation and erythropoietin-induced reticulocytosis in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1975. doi:10.1158/1538-7445.AM2011-1975
Abstract 3927: Characterization of galectin 3 as a putative BRCA1/BARD1 ubiquitin E3 ligase substrate
Authors: Carvalho Renato S. , Costa Thales N. , Sweet Michael , Monteiro Alvaro NA , Carvalho Marcelo A.
Published: August 2014

BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid screen as a binding partner of BRCA1. The functional heterodimer BRCA1/BARD1 is required for several of the cellular and tumor-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair pathway, transcription, cell cycle control, and ubiquitination. BARD1 also contains two tandem BRCT repeats at its C-terminus, a region shown to bind serine-phosphorylated peptides in vitro. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a yeast-two-hybrid screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for galectin 3 C-terminal region, residues 170-250 (OMIM 153619). Galectin 3 is a galactoside binding protein involved in several cellular processes including apoptosis control and RNA splicing. Moreover there are evidences that galectin 3 variants are correlated with tumor development and progression in breast and thyroid cancer. We confirmed galectin 3/BARD1 interaction by conducting co-immuneprecipitation analysis of ectopically expressed BARD1 and galectin-3 in HEK293T cells. The interaction between endogenous galectin 3 and BARD1 were validated in HeLa nuclear extracts. Using the same approach we co-immuneprecipitated BRCA1 and galectin-3, suggesting the presence of BRCA1, BARD1 and galectin-3 in the same complex. Interestingly the galectin 3 observed in this complex corresponds to a mono-ubiquitinated form, corroborating the idea that galectin 3 is a putative natural target of BRCA1-BARD1 heterodimer E3 ubiquitin ligase.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3927. doi:10.1158/1538-7445.AM2011-3927
Abstract 3929: ADA3, a component of ATAC complex regulates DNA damage response
Authors: Mirza Sameer , Mohibi Shakur , Gurumurthy Channabasavaiah B. , Katafiasz Bryan J J. , Zhao Xiangshan , Bele Aditya , Band Hamid , Band Vimla
Published: August 2014

Our long term research goal is to understand the biochemical mechanism of immortalization of human epithelial cells that represents early step in cancer progression. Over the years, we have generated several epithelial cell immortalization models, using viral oncogenes, mutated cancer genes, as well as radiation treatment of cells. In one of such immortalization model, we identified alteration/deficiency in activation (ADA3) protein as a novel human papilloma virus E6 oncoprotein binding protein, and as a regulator of p53 function. We and others have shown that human ADA3 protein is an essential component of various Histone acetyl transferase complexes (HATs) and thus controls transactivation of various transcriptional factors.

To gain insight into the physiological importance of ADA3, we established a conditional knockout mouse model and showed that loss of ADA3 led to embryonic lethality at an early stage of development. Using ADA3lox/lox mouse embryonic fibroblast (MEF) cell lines, we further showed that ADA3 plays an important role in DNA damage and repair pathway. Adenovirus cre-mediated deletion of ADA3 led to a global reduction of histone acetylation, severe G2/M cell cycle arrest, various aberration in nuclear structures, and massive chromosome aberration. Significantly, cells lacking ADA3 showed defects in recruitment of DNA repair associated proteins, BRCA1 and 53BP1 to the DNA damage foci. These results implicate ADA3 as a major cell cycle and DNA repair response regulating protein. These findings in the context of oncogenesis and chemotherapy will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2011-3929
Abstract 3936: Identifying molecular subgroups of gliomas in formalin fixed paraffin embedded material
Authors: Gravendeel Lonneke A. , de Rooi Johan J. , Erdem Lale , Eilers Paul H. , Kros Johan M. , Sillevis Smitt Peter A. , van den Bent Martin J. , French Pim J.
Published: August 2014

Gliomas are the most common primary brain tumors with heterogeneous morphology and variable prognosis. Histological classification, combined with the patients’ prognostic features, often guides treatment decisions. Unfortunately, differences in histology are subtle and therefore, diagnosis is subject to a large interobserver variability. To improve classification, we did expression profiling on fresh frozen tumor material of 276 glioma samples of all histological subtypes. This resulted in seven molecular subgroups, which correlated significantly better with survival than histology.

When validated in prospective studies these molecular clusters could contribute to clinical decision making. However, there is a lack of fresh frozen glioma material, and until now clinical studies have been performed on formalin fixed paraffin embedded (FFPE) material. Therefore, we would like to see whether our molecular clusters are reproducible in FFPE material.

Expression profiling was performed on 57 paired snap-frozen/FFPE glioma samples of all histological and molecular subtypes and three non-diseased brain samples. We collected FFPE material from the same patients that were included in our previous study (Gravendeel et al. Cancer Res 2009). FFPE expression profiling was performed using Hu_Ex_1.0_st “exon” arrays (Affymetrix) in combination with Nugen WT-Ovation technology (FFPE V2 and Exon modules).

FFPE expression profiles were assigned to a molecular cluster based on its nearest centroid using the 20.000 most variably expressed exons.

Preliminary analysis indicates that approximately 75% of all samples were assigned to the correct molecular cluster. Survival data confirmed that the molecular clusters identified using FFPE material retained significant prognostic value, similar to those obtained using fresh frozen material (p=0.0016).

Our data indicate that exon arrays in combination with Nugen WT technology are a suitable platform to perform expression profiling on FFPE samples. We are currently expanding our dataset to include FFPE samples from a large phase III European trial.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3936. doi:10.1158/1538-7445.AM2011-3936
Abstract 3955: Epigenetic silencing of microRNA-34b/c plays a pivotal role in pathogenesis of small-cell lung cancers
Authors: Tanaka Norimitsu , Toyooka Shinichi , Soh Junichi , Maki Yuho , Muraoka Takayuki , Ueno Tsuyoshi , Aokage Keiju , Asano Hiroaki , Tsukuda Kazunori , Yamane Masaomi , Oto Takahiro , Miyoshi Shinichiro
Published: August 2014

Background: Lung cancer is the leading cause of cancer-related death in the world. Small-cell lung cancer (SCLC) accounts for approximately 13% of lung cancers. Although chemotherapy results in initially high response rates, survival of patients with extensive stage disease is dismal. MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes that regulate cell proliferation and apoptosis. Recent studies have shown that, in cancer cells, expression of some miRNAs is silenced with DNA methylation in promoter region. Aberrant methylation of miR-34b/c has been reported in human malignancies, but not in SCLCs, causing up-regulation of oncogenes. In this study, we investigated the methylation and expression status of the miR-34b/c and the anti-proliferative effect by induction of miR-34b/c in SCLCs.

Methods: We examined 11 SCLC cell lines and 6 resected primary SCLC tumors (one tumor treated with preoperative chemotherapy and five without preoperative chemotherapy). Methylation and expression status of miR-34b/c were determined by methylation-specific PCR and quantitative PCR, respectively. Colony formation assay was applied to examine the anti-proliferative effect by induction of miR-34b/c and scramble sequence control with plasmid vector. The ability of cell migration was examined using cell migration assay.

Results: Aberrant methylation of miR-34b/c was found in 7 (64%) of 11 SCLC cell lines and 3 (50%) of 6 primary SCLC tumors. Expression of miR-34b/c in all methylation cell lines was silenced and restored with 5-aza-2’-deoxycytidine treatment, indicating that methylation caused gene silencing. Ectopic induction of miR-34b/c gene in SCLC cell line (H1048) showed significant inhibition of colony formation compared with induction of control vector, demonstrating anti-proliferative effect of miR-34b/c on SCLC. The cell migration was also significantly inhibited by induction of miR-34b/c in SCLC cell line tested.

Conclusion: Our results demonstrated that miR-34b/c methylation is frequent in SCLCs and induction of miR-34b/c inhibits the cell proliferation and migration, resulting that miR-34b/c silencing plays an important role in pathogenesis of SCLCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3955. doi:10.1158/1538-7445.AM2011-3955
Abstract 3956: Differential miR-141 expression in primary cultured human breast cancer epithelium (PCHBCEs) and normal adjacent part epithelium (PCHBNEs) correlates with tumor suppressor protein tyrosine phosphataseγ (PTPγ)
Authors: Lin Shu-Hong , Jiang Jinmai , Kuo Chieh-Ti , Ye Weiping , Xu Pingping , Li Hong , Liu Jie-Yu , Zhong Saiyi , Schmittgen Thomas D. , Lin Young C.
Published: August 2014

MicroRNAs (miR) have been shown to be extensively involved in tumorigenesis by post-transcriptional inhibition of oncogenes and/or tumor-suppressor genes. The purpose of our study is to investigate the difference in miRNA expression between cancer epithelium and epithelium from normal breast adjacent tissues. The clinical samples were procured from the Tissue Procurement Program at the Ohio State University Comprehensive Cancer Hospital from breast cancer patients who underwent partial or complete mastectomy. The miRNA profiling for one pair of PCHBCEC and PCHBNEC from the same patient was carried out, and the difference of miRNA expression and potential target genes were further verified by realtime qPCR in 7 pairs of clinical samples. The miRNA profiling showed that 24 miRNAs (let-7a, let-7c, let-7f, let-7g, miR-24, miR-28, miR-29a, miR-29c, miR-30a-5p, miR-30d, miR-92, miR-125a, miR-126*, miR-132, miR-135a, miR-135b, miR-137, miR-141, miR-182, miR-200c, miR-339, miR-365, miR-425-5p, miR-391, p<0.05) are statistically up-regulated in cancer while only 1 miRNA (miR-221) is down-regulated. Based on the magnitude of change and predicted target, we selected miR-141 for further validation. Compared with its normal adjacent counterpart, 4 PCHBCECs had lower miR-141 while 2 were up-regulated and 1 unaltered. Further validation on gene expression of the samples confirmed the negative correlation of miR-141 with its putative target PTPγ. Our comparison of PCHBCECs and PCHBNECs under the same genetic background demonstrated a distinct expression of miRNAs. The dysregulation of miR-141 was shown to result in modulation of the potential tumor-suppressor gene PTPγ which might have an impact on the etiological process of tumor lesion and discriminate cancer epithelial cells from their surrounding normal breast epithelial compartments. Our results implicate that miR-141 might serve as molecular biomarker for therapy of human breast cancer patients. (Supported by NIH R01 Grant ES 015212).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3956. doi:10.1158/1538-7445.AM2011-3956
Abstract 3961: Sec23A is a regulative target of miR-200c and miR-375 in prostate cancer and influences cell growth in vitro
Authors: Nolte Elke , Szczyrba Jaroslaw , Wach Sven , Jung Volker , Kremmer Elisabeth , Graesser Friedrich , Wullich Bernd
Published: August 2014

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. Using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs and to analyze the physiological consequences of miRNA deregulation using cell culture models.

Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries and microarray analyses. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. The impact of identified target genes on cell growth was examined by a standardized wound healing assay.

Results: Two of the miRNAs consistently deregulated in primary prostate cancer samples were miR-200c and miR-375 which were upregulated more than 1.5-fold in deep sequencing and 2.9-fold and 3.9-fold, respectively, in microarray experiments (p<0.001, ANOVA). Independent target prediction databases (including miRanda, Targetscan and PicTar) indicated that the 3’ untranslated region (3’UTR) of the Sec23a gene is a regulative target for both miR-200c and miR-375. This prediction could be confirmed using a luciferase reporter gene construct containing the Sec23a 3’UTR. Co-expression of the reporter gene construct with either miR-200c or miR-375 resulted in a reduction of reporter gene activity while mutation of the miRNA binding sites caused a loss of responsiveness to the respective miRNA. Furthermore we could show that transient expression of miR-200c and miR-375 reduced the amount of Sec23a protein in the HEK293 cell line. A transient knock-down of Sec23a in DU145 prostate cancer cells applying siRNA technique led to acceleration of wound healing and transient overexpression of Sec23a to a deceleration over a time period of 30 hours. In Western blot analyses the majority of primary tissue samples displayed a reduced amount of Sec23a protein.

Conclusion: Our findings demonstrate that the 3’-UTR of Sec23a is one regulative target of miR-200c and miR-375, which are commonly deregulated in prostate cancer. Sec23a is reduced in the majority of tumor samples analyzed and has a vital impact on cell growth as determined by wound healing experiments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3961. doi:10.1158/1538-7445.AM2011-3961
Abstract 3962: Knocked down miR-221/222 inhibits Akt pathway to suppress malignant glioma cell growth independent of PTEN status
Authors: Zhang Chunzhi , Zhang Anling , Hao Jianwei , Zhang Junxia , Jia Zhifan , Wang Guangxiu , Pu Peiyu , Kang Chunsheng
Published: August 2014

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that regulate gene expression after transcription. By base-pairing with the complementary sites in the 3’untranslated region (3’UTR) of the mRNA, miRNAs control mRNA stability and translation efficiency. Aberrant expression of miRNAs have been shown to be involved in tumorigenesis. MiR-221 and miR-222 were frequently overexpressed in human malignant tumor, including glioblastoma. By Northern Blot assay, we found that miR-221 and miR-222 upregulated in glioma cell lines. In addition, Akt pathway is aberrant activity in glioblastoma. Our work have proved that miR-221/222 promote malignant progression of glioma cell through activation of the Akt pathway. Once Akt activated, it promotes cellular proliferation and invasion ability and reduces cell apoptosis and radiosensitivity. By bioinformation analysis, we found that PTEN is a target of miR-221 and miR-222. By luciferase report assay, we identified that PTEN is a target of miR-221 and miR-222. It is unknown whether miR-221/222 activate Akt pathway dependent of PTEN status or not. We knocked down miR-221/222 with a specific antisense oligonucleotide and found that phenotype of glioblastoma cell changed in vitro in U251 (PTEN mutant) and LN229 (PTEN wild type), including increasing cell proliferation and invasion ability, reducing cell apoptosis and radiosensitivity. By Western Blot, knocked down miR-221/222-cells showed a decreased expression of EGFR, activated Akt, cyclin D, and Bcl-2. Furthermore, knockeddown miR-221/222 considerably decreased tumor growth in U251 cell xenograft model. By immunofluorescence assay, it was found that antisense-miR-221/222 significantly knocked down miR-221 and miR-222 in vivo. By Tunnel assay, antisense-miR-221/222 significantly increased cell apoptosis in vivo. By immunohistochemistry staining, some protein expression reduced, including expression of EGFR, activated Akt, cyclin D, and Bcl-2. Although miR-221 /222 is known to regulate PTEN and downregulation of miR-221/222 led to increased PTEN expression endogenously, the GBM suppressor effect of antisense-miR-221/222 is most likely independent of PTEN regulation because U251 has mutant PTEN. Taken together, our studies provide evidence that miR-221/222 may serve as a novel therapeutic target for malignant gliomas independent of PTEN status.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3962. doi:10.1158/1538-7445.AM2011-3962
Abstract 4056: Fluorescence labeling of different live cell populations and subsequent analysis by flow and imaging cytometry
Authors: Chan Leo L. , Alvarez Mark , Shen Dee , Lin Bo , Patton Wayne F.
Published: August 2014

Relative to conventional antibody-based immunohistological methods employing fixed and permeabilized cells, live cell imaging has presented challenges due to the lack of availability of compatible fluorescent probes. Additionally, instrument-dependent complications arise, such as the requirement that adherent cells be detached into a single cell suspension for flow cytometry, which can impact physiological response. A novel noncovalent fluorescence, long-term labeling technology was employed to stably incorporate green, red, or mixtures of green and red fluorescent dyes into the plasma membranes of different live cell populations. The dyes were well retained for up to 96 hours, faithfully transferred to daughter cells upon mitosis, yet displayed no tendency for nonspecific transfer to adjacent unlabeled cell populations. Since the described dyes do not covalently modify proteins within the cells, normal physiological responses are well preserved. Labeled cell populations were analyzed by conventional flow cytometry as well as with a novel imaging cytometer, the Cellometer Vision, incorporating inexpensive disposable counting chambers. Using a combination of bright-field and fluorescent images, the imaging system automates cell image acquisition and processing using novel counting algorithms for accurate and consistent measurement of cell concentration, fluorescent intensities and percentages of fluorescent positive cells. Results achieved using the imaging cytometer showed excellent correlation with those achieved by flow cytometry, but required 25-fold lower sample per analysis. As many as four cell subpopulations could be determined simultaneously. The described workflow provides a powerful, yet economic, approach applicable to long-term cell viability, cytotoxicity, cell adhesion, cell migration, lineage tracing and cell-cell fusion analysis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4056. doi:10.1158/1538-7445.AM2011-4056
Abstract 4057: Mono-ubiquitination of Smad2/3 by Smurf2 regulates TGF-β transcriptional response
Authors: Tang Liu-Ya , Yamashita Motozo , Tang Yi , Zhang Ying E.
Published: August 2014

Transforming growth factor-beta (TGF-β) regulates a wide array of biological processes ranging from proliferation to apoptosis, and alterations in its signalling pathway are associated with a variety of human diseases, including cancer. Through Smad proteins, TGF-β signal can be propagated into nuclear transcription machinery. Thus, fine tuning of Smads activity is crucial for TGF-β signal transdction. Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase and previous data shows that Smurf2 negatively regulates TGF-β signaling by inducing polyubiqutination and degradation of Smad proteins. Here, we find a different type of ubiquitination – mono-ubiquitination of Smad2/3 by Smurf2. Smad2/3-Smurf2 interaction and Smad2/3's mono-ubiquitination by Smurf2 are enhanced by TGF-β induced linker phosphorylation. The ubiquitination depends on Smad2/3's PY motif and Smurf2's E3 ligase activity. Cycloheximide treatment assay revealed that this ubiquitination has no effect on Smad2/3 protein stability, but the transcriptional activity is inhibited as shown by both luciferase assay and real-time PCR analysis. Thus, our findings reveal a novel regulatory role of Smurf2 in TGF-β signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4057. doi:10.1158/1538-7445.AM2011-4057