bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3616: Highly potent antibody-amanitin conjugates cause tumor-selective apoptosis
Authors: Anderl Jan , Müller Christoph , Heckl-Östreicher Brigitte , Wehr Roland
Published: June 2012

Alpha-amanitin blocks RNA polymerase II activity and is the most specific and most potent inhibitor of eukaryotic transcription. While widely used as a tool in molecular biology, only a few attempts have been made to employ alpha-amanitin as a therapeutic agent in oncology. The reason for this may be the high liver toxicity caused by the transport of amanitin into hepatocytes by the organic-anion transporter 1B3 (OATP1B3) present on hepatocytes, but not on other cell types. In this study we show that a direct impact on the transcription machinery can provide a novel approach in tumor therapy, provided that the amanitin can be targeted to tumor cells. This was achieved by using tumor-specific antibodies of high affinity armed with amanitin via suitable linker moieties.

Using the HER2 specific monoclonal antibody Herceptin (Her) as a carrier, the antibody-drug conjugate (ADC) Her-DSC-30.0134 exhibits antiproliferative activity in HER2-expressing human tumor cell lines like SK-OV-3, SK-BR-3 and NCI-N87 in picomolar concentrations, indicating an up to 100.000-fold higher toxicity of the conjugate over non-conjugated alpha-amanitin. On cells lacking the HER2 receptor, e.g. HEK293 cells, toxicity of Her-DSC-30.0134 was not detected up to micromolar concentrations. This is also observed in HEK293 cells transfected with OATP1B3 (which incorporate amanitin similar as hepatocytes) indicating that amanitin-antibody-conjugates are no longer substrates for OATP1B3. These data suggest that a low hepatotoxicity of amanitin conjugates is expected.

Subsequently, antitumoral activity of Her-DSC-30.0134 was evaluated in vivo in a s.c. xenograft mouse model using Herceptin-sensitive SK-OV-3 and Herceptin-resistant JIMT-1 cells. The ADC is well tolerated in nude mice and shows complete tumor remission in 7 out of 7 animals after a single i.v. injection in the SK-OV-3 and JIMT-1 models. By contrast, Herceptin shows only moderate antitumoral activity in the SK-OV-3 and no activity in the resistant JIMT-1 model, even after multiple injections. These data encourages further examination of amanitin-based antibody conjugates as highly potent and selective compounds for the treatment of various maligancies.

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 3616. doi:10.1158/1538-7445.AM2011-3616
Abstract 3666: Inhibition of the tumor growth and prevention of metastases with MORAb-004, a human monoclonal IgG targeting endosialin/TEM-1
Authors: Tomkowicz Brian E. , Skidan Igor , Rybinski Katherine , Lin JianMin , Grasso Luigi , Nicolaides Nicholas , Sass Philip , Zhou Yuhong
Published: June 2012

Endosialin/Tumor Endothelial Marker-1 (TEM-1) is a cell surface glycoprotein that is expressed on cells involved in the development of tumor vasculature, primarily pericytes, smooth muscle, and stromal fibroblasts. Studies have found endosialin to play a key role in tumor growth and neovessel formation in numerous cancer types including (but not limited to) renal, breast, colon, pancreatic, lung, endometrial, ovarian, melanoma, sarcoma, and neuroectodermal tumors. We are currently evaluating the therapeutic efficacy of MORAb-004, a human monoclonal IgG1 targeting TEM-1, in a Phase I clinical trial. Because MORAb-004 only recognizes human/monkey TEM-1 and has no cross-reactivity with rodent TEM-1, we generated a human TEM-1 knock-in mouse line in order to assess its activity in in vivo tumor models. In a B16-F10 subcutaneous melanoma model we found that MORAb-004 significantly inhibited tumor progression by ∼70%. Moreover, using the Lewis Lung Carcinoma model of metastatic disease our results showed that MORAb-004 had a significant inhibitory effect on the number of lung metastases. Taken together our results demonstrated the ability of MORAb-004 in inhibiting the growth of solid tumors and preventing the emergence of distant metastases. The potential MOA is currently under evaluation and 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 3666. doi:10.1158/1538-7445.AM2011-3666
Abstract 3682: Response of biomarkers of inflammation (IL-8,TNF-α, sTNFRI & II) to cruciferous vegetable feeding in a controlled diet study in humans: Effects of GSTM1 and GSTT1 genotypes
Authors: Navarro Sandi L. , Schwarz Yvonne , Song Xiaoling , Wang Ching-Yun , Chen Chu , Lampe Johanna W.
Published: June 2012

Isothiocyanates (ITC) in cruciferous vegetables modulate several signaling pathways involved in carcinogenesis, including nuclear factor kappa-B (NF-κB), a transcription factor and central mediator in the generation of inflammation. Glutathione S-transferase (GST)M1 & GSTT1 metabolize ITC; genotypic differences in biologic response to cruciferous vegetable intake, and variation in ITC pharmacokinetics have been reported. Previously, we found reduced serum IL-6 and CRP concentrations in response to cruciferous (broccoli family) and apiaceous (carrot family) vegetable feeding compared to a basal diet in a randomized, crossover trial. Reductions were most marked among individuals with a GSTM1-null genotype. Here, we extend our panel of biomarkers in this intervention to include serum interleukin (IL)-8, tumor necrosis factor-alpha (TNF-α), and soluble TNF receptors I & II (sTNFRI & II). We tested whether supplementation of cruciferous or apiaceous vegetables alters serum concentrations of these markers, and whether this response is GSTM1/GSTT1 genotype-dependent. Men (n=26) and women (n=30), 20-40 y, ate four 14-day controlled diets – basal (fruit &vegetable-free), basal + two doses of crucifers (single & double “dose”), and single-dose cruciferous + apiaceous vegetables – fed per kg body weight. Fasting bloods from days 0 & 14 of each period were analyzed for serum inflammatory markers (IL-8 & TNF-a using the High Sensitivity Human Cytokine Panel, sTNFRs using the Human Soluble Cytokine Receptor Panel, Millipore). In this healthy population, we observed no overall reduction in any of the current biomarkers in response to vegetable supplementation. However, similarly to what we observed with IL-6 and CRP, both sTNFRI & II were lower at day 14 on the double-dose cruciferous vegetable diet among GSTM1-null individuals (P=0.006 & 0.007, respectively). NF-kB is one regulator of these inflammatory markers; but overlap with other pro-inflammatory pathways that may be differently regulated by diet, e.g., STAT3, may preclude observation of an effect. Such diet interventions may be more effective in populations with higher circulating inflammation biomarkers.

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 3682. doi:10.1158/1538-7445.AM2011-3682
Abstract 3710: Individual and combined effects of Tamoxifen and fish oil on mammary carcinogenesis in polyoma middle T transgenic mice
Authors: Manni Andrea , Xu Haifang , Washington Sharlene , Aliaga Cesar , Cooper Timothy K. , Richie John P. , Prokopczyk Bogdan , Calcagnotto Ana , Trushin Neil , Hamilton Christopher , Demers Laurence M. , Liao Jason , Verderame Michael F. , El-Bayoumy Karam
Published: June 2012

There are currently no known effective regiments able to prevent the development of estrogen-receptor (ER) negative breast cancer in humans. We hypothesize that ER, frequently upregulated in preneoplastic mammary lesions, may contribute to the development of hormone-independent tumors through crosstalk with multiple cellular oncogenic pathways. Therefore, a multi-targeted approach is likely to be necessary to prevent ER negative tumors. In our previous study, we tested this hypothesis in CD rats and we showed that omega-3 rich fish oil (FO) potentiated the suppressive effect of Tamoxifen (Tam) on mammary carcinogenesis (A. Manni, et al. Cancer Prev Res 3:322, 2010). In the present investigations, we tested this hypothesis in polyoma middle T transgenic mice, since in this experimental system the development of ER negative tumors is preceded by preneoplastic lesions overexpressing functional ER, as evidenced by the concomitant upregulation of progesterone receptors which are products of estrogen action. Administration of increasing concentrations of Tam (1 ppm, 10 ppm, and 100 ppm) admixed with 20% corn oil (CO) modified AIN-76A diet, starting at 3 weeks of age, significantly delayed mammary carcinogenesis, inhibited tumor multiplicity by ∼50%, tumor volume by ∼90%, and tumor weight by ∼70% in a dose dependent fashion. At termination (week 12), however, tumor incidence was nearly 100% in all experimental groups. When Tam treatment was started after tumors had developed, no inhibition of tumor growth was observed, a finding consistent with the absence of ER in invasive adenocarcinomas in this experimental model. Administration of increasing concentrations of FO in the diet (5% FO + 15% CO; 10% FO + 10% CO, 17% FO + 3% CO) started at 3 weeks of age did not affect any of the tumor parameters compared to the control 20% CO diet. Combined administration of suboptimal (1 ppm) and optimal (100 ppm) doses of Tam with 17% FO and 10% FO respectively, delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as observed with Tam alone. Therefore, the results indicate that, under these experimental conditions, FO failed to potentiate the chemopreventive action of Tam. Our results, however, provide support for the role of ER expression by the preneoplastic lesions in the development of hormone independent tumors and, consequently, for the importance of including ER targeting in combined chemoprevention strategies.

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 3710. doi:10.1158/1538-7445.AM2011-3710
Abstract 3757: PTGS1 and PTGS2 polymorphisms, fatty acid intake, and risk of colon and rectal cancer
Authors: Habermann Nina , Slattery Martha L. , Poole Elizabeth M. , Xiao Liren , Galbraith Rachel L. , Duggan David , Kulmacz Richard J. , Caan Bette J. , Koepl Lisel , Coghill Anna E. , Makar Karen W. , Lundgreen Abbie , Potter John D. , Ulrich Cornelia M.
Published: June 2012

Introduction: Inflammation plays a major role in colorectal cancer. COX-1 and COX-2 convert n-3 and n-6 fatty acids into prostaglandins which have anti- and pro-inflammatory effects, respectively. We examined potential interactions between polymorphisms in PTGS1 (COX-1) and PTGS2 (COX-2) and fat and fatty acid (FA) intake in relation to colon and rectal cancer risk.

Methods: A linkage-disequilibrium-based tagSNP-selection algorithm (r2=0.90, MAF=4%) identified tagSNPs in PTGS1 and PTGS2. We genotyped 16 SNPs in PTGS1 and 17 SNPs in PTGS2 on the same Illumina platform in two independent study populations (colon cancer: 1502 cases, 1862 controls; rectal cancer 713 cases, 914 controls). We investigated potential interactions with intake of total fat, saturated FA (SFA), monounsaturated FA (MUFA), polyunsaturated FA (PUFA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) on risk of colon or rectal cancer.

Results: We observed several statistically significant interactions between PTGS1 and PTGS2 genotypes and fat and FA intake (Table). However, few interactions were consistent across fat variables and fell into three general categories of associations: a) individuals with the variant allele and low intake of total fat, SFA, or MUFA were at increased risk for rectal cancer (PTGS1 rs10306122-G, p int=0.01-0.01; PTGS2 rs4648268-A; p int=0.01-0.02); b) individuals with the variant allele and high EPA and DHA intake were less likely to develop colon cancer (PTGS1 rs10306110-G, p int=0.003-0.006); and c) individuals with the wildtype genotype and high EPA and DHA intake had stronger protection from colon cancer (PTGS2 rs4648310-A, p int=0.01-0.05). No interactions were observed for AA.

Conclusion: Our study provides some evidence that genetic variability in PTGS1 and PTGS2 and intake of total fat, SFA, or MUFA and EPA or DHA may interact in causing rectal and colon cancer, respectively.

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 3757. doi:10.1158/1538-7445.AM2011-3757
Abstract 3889: Direct evidence that the MRN complex is required for a pathway that processes Top2 covalent complexes
Authors: Soans Eroica , Mishina Margarita , Nitiss John L.
Published: June 2012

Drugs targeting topoisomerase II (Top2) such as etoposide or doxorubicin generate enzyme mediated DNA damage. During the normal Top2 reaction, the enzyme forms a covalent enzyme DNA intermediate consisting of a 5’ phosphotyrosyl linkage between the enzyme and DNA. Many compounds that generate Top2 mediated damage inhibit the enzyme-catalyzed religation step leading to DNA breaks. Therefore an important aspect of the repair of Top2 mediated damage includes removal of the protein covalently bound to DNA. One way of eliminating that protein is by proteolysis. Liu and colleagues have clearly demonstrated that proteolysis plays a role in repairing Top2 beta damage1 and it is likely that proteolysis is also important for repairing Top2 alpha mediated damage. Proteolysis cannot remove all of the peptide bound to DNA; therefore nucleolytic enzymes are also needed for repairing Top2 damage. Since the MRN complex is required for nucleolytic removal of the meiotic Top2-like protein Spo11, it has been hypothesized that the MRN complex also participates in removal of Top2 protein from DNA following cellular treatment with Top2 poisons. However, there is no direct evidence for processing of Top2 mediated damage by the MRN complex. We have used the ICE assay to directly measure Top2 covalent complexes in mammalian cells treated with Top2 poisons such as etoposide. The ICE assay uses CSCl centrifugation to isolate protein:DNA covalent complexes. We have combined this assay with siRNA knockdowns to assess whether specific protein complexes are required for processing Top2 covalent complexes. Since this assay depends on recognition of Top2 epitopes by monoclonal antibodies directed towards Top2 isoforms, we have examined levels of Top2 complexes in cells treated with the proteasome inhibitor, MG132. In cells treated with siRNA directed against Mre11, we observed an increase in Top2:DNA covalent complexes following treatment with either etoposide or mAMSA. These results provide direct evidence that the MRN complex is required for at least one pathway for removal of Top2 covalent complexes. It is likely that this MRN dependent pathway is capable of removing Top2 bound to DNA without proteolytic processing of the covalent complex. We are currently using this approach to assess other recently described processing pathways including pathways requiring CtiP and pathways requiring Tdp2.

(1) Mao, Y.; Desai, S. D.; Ting, C. Y.; Hwang, J.; Liu, L. F. J Biol Chem 2001, 276, 40652.

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 3889. doi:10.1158/1538-7445.AM2011-3889
Abstract 3900: Formaldehyde induces DNA damage and BRCA1 nuclear export
Authors: Ponce Esmeralda M. , Louie Maggie C.
Published: June 2012

Formaldehyde is a colorless, potent, flammable gas, commonly used to manufacture building materials and other household and personal care products. Most living organisms are exposed to some levels of formaldehyde in one way or another. Studies have shown formaldehyde to have mutagenic and genotoxic effects on proliferating cells. In order to understand the effects of formaldehyde on cancer progression, we examined the effects of formaldehyde on estrogen receptor positive breast cancer cells, MCF-7. Our data suggest that low concentrations of formaldehyde decrease the proliferation of MCF-7 cells. Further analysis at the molecular level suggests that formaldehyde increases the expressions of p53 and BRCA1, two tumor suppressor genes known to play important roles in DNA repair. Consistent with previous reports that high concentrations of formaldehyde results in irreversible DNA damage, our results suggest that exposure to low concentrations of formaldehyde is sufficient to induce DNA damage. Protein and gene expression analyses suggest that p53 and BRCA1 expression levels are dependent on the concentration of formaldehyde and length of exposure. Additionally, our data show that BRCA1 undergoes nuclear export in response to formaldehyde which is consistent with its role in DNA damage surveillance and repair.

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 3900. doi:10.1158/1538-7445.AM2011-3900
Abstract 3904: Array CGH analysis of cervical cancer
Authors: Zuna Rosemary E. , Aston Christopher E.
Published: June 2012

Introduction: We hypothesize that there are characteristic chromosomal gains and losses in invasive cervical cancers that correlate with histologic cell type and HPV genotype.

Methods and Materials: Array CGH analysis was performed on DNA extracted from 39 formalin-fixed, paraffin embedded invasive cervical cancer cases using 1.4MB BAC microarray chips (HumArray3.2, UCSF Comprehensive Cancer Center Array Core). Significant gains and losses were determined looking for convergent results from a random permutation approach based on GISTIC (Genomic Identification of Significant Targets in Cancer, Beroukhim R et al: PNAS 104: 20007, 2007), a modification of this approach using weighted sums over neighboring loci, frequency of gains and losses at each locus, and t-tests comparing log2 ratios from the cases to those from four control samples from non-cervical cancer tissue. The log2 ratios derived for each of the 2464 individual loci were aggregated for each locus over the entire set of 39 cases and for defined subsets including histologic subtype (squamous [SC:N=19] cancers, adenocarcinomas [AD:12], adenosquamous carcinomas [AS:6]) and HPV genotype [HPV16:12 and HPV18:14].

Results: Significant gains (108 individual loci) and losses (110 loci) were identified in the overall population. Congregations of significant gains were found on chromosomes 3q (8 loci), 1q (7), 1p (5) and losses on 4q (8 loci), 11q, (6) 4p (5), 3p (5). Overall,17 individual loci were significantly increased and 19 were decreased across the overall population as well as for each of the histologic subsets suggesting associations with cervical cancer per se. Individual loci with the most significant gains (P<0.001) were 3q26.3-3q27 (gained in 95% of cases), 19p13.2 (98%), 1q42-43 (93%) and losses (P<0.001) were 4q22 (100%), 11q22 (100%), 3p14 (90%) and 7p12.1 (100%). Differential changes associated with histologic cell type (P<0.001) included multiple gains in 1p (SC), 20q (AC), and losses in 3p (SC), 4p (AS). Multiple gains in 1p and 3q were related to HPV16 lesions while gains in 5p and 19q were related to HPV18. The HPV associations were somewhat influenced by cell type: 8/9 (89%) of AD (3 untyped) were HPV16, but only 2/18 (11%) of SC (1 untyped) were HPV16.

Conclusions: Characteristic chromosome copy number alterations for regions and individual loci were identified for cervical cancer cases including patterns for different histologic subtypes and HPV genotypes. The regions harboring these loci can potentially be developed for screening, assessment of prognosis and therapeutic intervention.

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 3904. doi:10.1158/1538-7445.AM2011-3904
Abstract 3966: Regulation of Chd5 expression by microRNA-211 and microRNA-130b in colorectal cancer
Authors: Cai Chunxiao , Ashktorab Hassan , Smoot Duane T. , Liu Yulan , Gu Xinbin
Published: June 2012

PURPOSE: Chromodomain helicase DNA-binding protein 5 (CHD5) is identified as a novel tumor suppressor in particular of colorectal tumorigenesis. There is limited information about the regulation of CHD5 expression. In this study, we investigated the role of microRNAs (miRNAs) in regulation of CHD5 protein expression in colorectal cancer. DESIGN METHODS: MiR-211 and miR-130b as predictive target CHD5 candidate miRNAs were selected through Targetscan software. A high level of CHD5 protein colorectal cancer HCT-116 cell line was used to establish miR-211 and miR-130b stable transfected cell lines (HCT-116miR-211 and HCT-116miR-130b) as test models, following by MTT, colony assay, flow cytometry for cell viability examination. Quantitative real-time PCR and Western blot analysis were also used for measure miR-211, miR-130b and CHD5 expression levels in HCT-116miR-211 and HCT-116miR-130b. CHD5-associated proteins expression levels were assessed as well. RESULTS: MiR-211 and miR-130b were cloned into lentiviral GFP expression miRNA vector, separately. Retrovirus was produced by using lentiphos TMHT packaging system and infected into HCT-116 cell line. The expression levels of miR-211 in HCT-116miR-211 and miR-130b in HCT-116miR-130b were significantly increased (miR-211 increased 16 times, and miR-130b increased 8 times) and at the same time CHD5 expression levels was decreased. Compared to the vector control cell lines, the levels of cell proliferation and colony formation were higher in HCT-116miR-211 and HCT-116miR-130b. In addition, the proportion of cell number in the G0-G1 phase of HCT-116miR-211 and HCT-116miR-130b was lower than those in the control cell lines. CONCLUSION: Up-regulation of miR-211 and miR-130b induced the malignant phenotype of HCT-116 cells via block of CHD5 expression. That implicated the miRNAs might be one of the mechanisms that lead CHD5 down-regulated in colorectal cancer. This work was supported in part by grants P20 CA118770 and U54 CA091431 from National Cancer Institute.

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 3966. doi:10.1158/1538-7445.AM2011-3966
Abstract 3976: The role of a novel oncomiR, miR-135b, in lung cancer metastasis and clinical application
Authors: Lin Ching-Wen , Lin Jau-Chen , Chang Yih-Leong , Chen Hsuan-Yu , Pan Szu-Hua , Hong Tse-Ming , Yang Pan-Chyr
Published: June 2012

microRNA(miR) may function endogenous siRNA and regulate a diversity of cellular function through translational repression. Dysregulations of miR expression are involved in carcinogenesis and progression in various cancers. By combining the miR microarray and genomic DNA-methylation array, we identified an intronic miR, miR-135b, which is up-regulated in high invasive lung cancer cell CL1-5. The promoter region of miR-135b is highly de-methylated in high invasive cells and its expression in the less invasive lung cancer cells can be induced by demethylating agent 5’-aza-CdR treatment. Ectopically expression of miR-135b enhanced cell invasive and migratory ability in vitro as well as the orthotopic tumor growth and lung metastasis activity in vivo, whereas specific inhibition of miR-135b decreased tumor growth and metastatic activities. Using the bioinformatic prediction tools, we identified and confirmed that miR-135b could directly repress the expression of Lzts1 through 3’UTR seed-binding. Ectopically-expressed Lzts1 inhibited cell invasion and migration activity, implicating that miR-135b may promote lung cancer cell metastasis by down-regulating Lzts1. Moreover, high level of miR-135b and low level of Lzts1 expressions were associated with poor overall survival in lung cancer patients. We also showed that the miR-135b expression was regulated by NF-κB signaling-cascade and synergistically induced by co-treatment of 5’-aza-CdR and TNF-α, implying that the abnormal expression of miR-135b in cancers may result from inflammatory stimulation and epigenetic modulation. Our results support that miR-135b is an oncogenic miR that may contribute to lung cancer progression and metastasis. The miR-135b/Lzts1 pathway is a promising therapeutic target of lung 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 3976. doi:10.1158/1538-7445.AM2011-3976
Abstract 4000: Trastuzumab targeting of HER2 upregulates miRNA-194 and downregulates profilin 2 and DNMT3A in HER2 positive breast cancer
Authors: Le Xiao-Feng , Wu Yun , Spizzo Riccardo , Yoo Suk-Young , Wang Jing , Calin George A. , Bast Robert C.
Published: June 2012

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of oncoprotein HER2, is widely used in standard therapy for women with HER2-positive breast cancer. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis, and apoptosis have been intensively investigated, the effect of trastuzumab on microRNA (miRNA) is unknown. Since miRNA provides a novel layer of gene regulation and may play role in trastuzumab action, we have consequently performed miRNA microarray profiling before and after trastuzumab treatment in both SKBr3 and BT474 breast cancer cells that overexpress HER2. Trastuzumab significantly decreased five human miRNAs and increased three others in SKBr3 cells, whereas in BT474 cells it significantly decreased two miRNAs and increased ten. The only miRNA that shared the same change in both cell lines was miRNA-194 (miR194), which was upregulated following trastuzumab treatment. Upregulation of miR194 by trastuzumab was further validated in vitro and in vivo by quantitative reverse transcription PCR (QRT-PCR) or Northern blotting. miR194 expression in 92 cases of breast cancer and 21 normal breast tissues (all were FFPE samples) was examined by QRT-PCR. miR194 levels were significantly higher in breast cancer samples versus normal tissues (p=0.0003), in estrogen-positive breast cancer samples (p=0.002), and in HER2-negative breast cancer samples (p=0.032). Importantly, expression of precursor miR194 specifically downregulated the protein levels of DNA methytransferase DNMT3A and actin cytoskeleton organizer profilin 2. miR-194 specifically inhibited the luciferase activity of wild type DNMT3A 3’-UTR construct, but not that of mutant vector, indicating that DNMT3A is a direct downstream target of miR-194. Both trastuzumab treatment and overexpression of miR-194 inhibited cell motility of breast cancer cells, which may result from the inhibition of DNMT3A and profilin 2. In summary, trastuzumab treatment upregulates miR194 expression and may exert its therapeutic effects through downregulation of miR194-downstream targets DNMT3A and profilin 2 in HER2-positive breast cancer cells.

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 4000. doi:10.1158/1538-7445.AM2011-4000
Abstract 4005: The leukemia-associated fusion protein DEK-NUP214 induces proliferation through an mTOR-dependent mechanism
Authors: Sandén Carl , Ageberg Malin , Gullberg Urban
Published: June 2012

Acute myeloid leukemia (AML) is characterized by the dysregulated proliferation and impaired differentiation of myeloid precursor cells. The majority of these leukemias harbor genetic translocations, which determine both the molecular mechanistics and the prognosis of the disease. The t(6;9)(p22;q34) chromosomal translocation is found in 1% of AML, where it is associated with young age and poor prognosis. The translocation occurs between specific introns in the gene DEK on chromosome 6 and the gene NUP214 on chromosome 9, creating the fusion gene DEK-NUP214. The role of DEK-NUP214 in leukemogenesis is still largely uncharacterized. To obtain an experimental model of the disease, we expressed the fusion gene in the myeloid cell line U937 and studied the phenotype of the stable clones. We show that cells expressing DEK-NUP214 proliferate faster and also sustain their proliferative capacity longer in culture than their normal counterparts. Cellular proliferation is regulated by a wide range of signaling pathways, but many converge on the activating phosphorylation of the mechanistic target of rapamycin (mTOR) at Ser2448. Western blot analysis of the clones revealed that cells expressing DEK-NUP214 have higher levels of both phosphorylated and total mTOR protein. To determine the effect on downstream cellular functions, we proceeded to study mTOR-dependent translation and metabolism. We performed a global translation assay where the incorporation of radioactively labeled amino acids into newly synthesized proteins reflects the rate of translation. The results show that cells expressing DEK-NUP214 have a markedly increased translation rate. Cellular metabolism was studied by measuring the consumption of glucose and the production of lactate in cell supernatant. We demonstrate that cells expressing DEK-NUP214 produce less lactate, despite equal glucose consumption and increased proliferation. In concordance with increased mTOR activity, our results suggest that cells expressing DEK-NUP214 shift their metabolism from glycolysis to oxidative phosphorylation. The increased activity of mTOR thus leads to translational and metabolic changes that could play a role in the leukemogenic effect of DEK-NUP214. Interestingly, proliferation induced by DEK-NUP214 is highly dependent on mTOR. Treatment with the mTOR inhibitor everolimus (RAD001) reduces the proliferation of the DEK-NUP214 cells to the level of the control cells, without affecting the control cells. This pivotal role of mTOR suggests that leukemias harboring the t(6;9)(p22;q34) translocation may be susceptible to treatment with either rapamycin or the novel mTOR inhibitors that are approaching the clinic.

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 4005. doi:10.1158/1538-7445.AM2011-4005
Abstract 4009: Regulation of mTOR pathway by the orphan nuclear receptor NR4A1 (TR3/Nur77) in non-small cell lung cancer: Role of TR3 in AMPK activation
Authors: Lee Syng-Ook , Andey Terrick , Jin Un-Ho , Kim Kyounghyun , Singh Mandip , Safe Stephen
Published: June 2012

The orphan nuclear receptor NR4A1 (TR3/Nur77) is overexpressed as a nuclear protein in multiple human tumors and this receptor exhibits pro-oncogenic activity in many tumor types. A recent study in this laboratory has shown that deactivation of TR3 decreases proliferation and induces apoptosis through inhibition of Sp1-dependent anti-apoptotic genes. In this study, we show that TR3 is overexpressed in human non-small cell lung cancer (NSCLC) tissues compared with adjacent normal lung tissues and high TR3 expression levels are correlated with shorter survival for patients with NSCLC (P = 0.0112). Treatment of NSCLC cells with small interfering RNAs for TR3 or the TR3 deactivator, 1,1-bis(3’-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), decreased cell proliferation and induced apoptosis through dual-targeting of mTOR pathway and Sp1 transcription factor. Knockdown of TR3 inhibited mTOR and its two major downstream targets, p70S6K and 4E-BP1 through LKB1-independent activation of AMPK but not through inactivation of AKT nor Sp1. We also observed that AMPK activation by TR3 knockdown is primarily due to induction of sestrin2 by enhancing p53-dependent transactivation activity. Furthermore, DIM-C-pPhOH (30 mg/kg/day) induced apoptosis and inhibited tumor growth in an orthotopic mouse model of human NSCLC, and decreased tumor number and size in a lung metastatic tumor model using tail vein injection. These results demonstrate for the first time that endogenous nuclear TR3 regulates AMPK-mediated mTOR pathway and TR3 deactivation represents a novel approach for treatment of NSCLC.

Grant support: National Institutes of Health (R01CA124998)

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 4009. doi:10.1158/1538-7445.AM2011-4009
Abstract 4387: Diversity Outbred: a new, highly diverse mouse stock for toxicology and cancer
Authors: Fancher Karen S. , Svenson Karen L. , Chesler Elissa J. , Churchill Gary A.
Published: June 2012

The Diversity Outbred (J:DO) stock is a new heterogeneous, highly diverse mouse resource designed for complex trait analysis and toxicology studies as well as to facilitate rapid mapping with single gene resolution. This stock was derived from crossing partially-inbred Collaborative Cross strains, which are a mixture of eight genetically well-defined JAX® Mice inbred strains, including three wild-derived. Subsequent maintenance follows a randomized outcross non-sibling mating scheme to retain maximum genetic diversity. Each Diversity Outbred mouse is genetically unique, representing a novel combination of alleles present in the founder strains. To determine the extent to which this genetic variation leads to phenotypic variation, over 50 metabolic measurements including plasma glucose, insulin, triglycerides, cholesterol, and body composition were collected, under both standard chow and high fat diet conditions. The Diversity Outbred animals displayed a wide spectrum of metabolic phenotypes, home cage nesting behavior and aggressiveness. Mouse-to-mouse variation is demonstrated by fold-differences in plasma triglycerides and creatine kinase levels, both as high as 11-12. Plasma glucose, free fatty acids, thyroxine, and percentage of fat all showed differences of 3-fold or greater. These fold-changes are comparable to the variation among unique inbred mouse strains, and mimic the diversity within human populations. The J:DO stock represents a new powerful tool for rapid gene mapping, toxicology/pharmacogenomic screens and compound evaluation for a wide variety of human diseases, including cancer and metabolic diseases.

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 4387. doi:10.1158/1538-7445.AM2011-4387
Abstract 4389: Copper and platinum uptake in cultured rat dorsal root ganglion neurons
Authors: Liu Johnson J. , Kim Yaeseul , Mercer Julian F.B. , McKeage Mark J.
Published: June 2012

Peripheral neuropathy is a dose-limiting toxicity of platinum drugs due to their accumulation in the dorsal root ganglia (DRG) and damage to sensory neurons. Our previous studies showed a specific pattern of expression of copper transporters in rat DRG tissue (Liu JJ et al 2009; Ip V et al 2010). Here we aimed to investigate their functional activity in the uptake of copper and platinum in cultured DRG neurons in vitro. Dissociated rat DRG neurons were cultured in N2-supplemented Neurobasal-A medium. Cellular metal levels were measured by the inductively coupled plasma mass spectrometry. The copper content of the cultured DRG neurons increased linearly with time (R2, 0.7952 ∼ 0.8903) during a one hour exposure to 3 ∼ 100 µM of CuCl2. The rate of cellular uptake of copper had nonlinear dependence (R2, 0.9629) on the exposure concentration of CuCl2, with a maximal uptake rate of 0.237 ± 0.041 nmole/min/mg protein and CuCl2 concentration achieving half-maximal uptake of 49.1 µM. The uptake of copper was temperature-dependent, stimulated by ascorbate and inhibited by zinc. CTR1 mRNA was detectable in cultured DRG neurons and its protein co-localised with microtubule-associated protein 2-positive neuronal cell bodies. Oxaliplatin inhibited copper uptake by DRG neurons and their platinum content increased linearly with time (R2, 0.916) during a one hour oxaliplatin exposure. In conclusion, CTR1 is functionally expressed by rat DRG neurons in primary culture. Oxaliplatin accumulates and inhibits copper uptake in DRG neurons. Supported by research grants from Cancer Society of New Zealand and McBeath Child Cancer Trust Fund.

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 4389. doi:10.1158/1538-7445.AM2011-4389
Abstract 4395: Functional domains in glyceraldehyde-3-phosphate dehydrogenase modulating its enzymatic activity and intracellular localization
Authors: Phadke Manali S. , Mishra Anurag , Krynetkaia Natalia , Krynetskiy Evgeny
Published: June 2012

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that plays a pivotal role in energy homeostasis of cancer cells. In response to several types of stress including genotoxic stress, GAPDH accumulates in the nucleus; its intranuclear functions remain to be elucidated. Earlier we demonstrated that, following genotoxic stress and intranuclear translocation in human lung carcinoma cells, GAPDH binds yet to be identified nuclear components. Using site mutagenesis, we prepared several mutated variants of GAPDH containing T99I and K259N amino acid substitutions, or a stop codon at position 263. Wild type GAPDH and mutated variants were expressed in BL21(DE3) bacterial system and purified by Ni-chromatography. Catalytic properties of the mutant proteins were compared with the wild type GAPDH. Catalytic activity of GAPDH protein was significantly reduced in the T99I mutant variant (Vmax 1.6±0.33 U/mg vs Vmax 40±1.9 U/mg of wild type GAPDH), while there was no significant difference in Vmax between the K259N variant and wild type GAPDH (p = 0.97). Truncated GAPDH (K263STOP) did not reveal any detectable catalytic activity. To assess the effects of mutations on intracellular localization of GAPDH, we prepared synthetic constructs coding for GFP-fusion variants of GAPDH (T99I, K259N, K263STOP) and used them for transient transfection of isogenic human carcinoma cell lines HCT116 (40-16; p53+/+ and 379.2; p53-/-) and SW48 (297; p53+/+ and 213; p53-/-). Intracellular distribution of wild type and mutant GAPDH was assessed by confocal microscopy and image analysis. Consistent with previous findings, wild-type GAPDH was excluded from the nucleus in the absence of stress, and accumulated in the nucleus following treatment with genotoxic agent cytarabine. In contrast, both T99I and K259N mutations significantly increased intranuclear content of GAPDH before and after stress. Truncated variant GAPDH (1-262) was evenly distributed in cytosolic and nuclear compartments. No significant difference was found in GAPDH localization between p53-proficient and p53-deficient isogenic cell lines indicating that p53 does not mediate GAPDH intranuclear localization. Our results identify the important functional domains of GAPDH that determine GAPDH intracellular localization. These domains are involved in interactions with intranuclear components of the cells experiencing genotoxic stress.

* both have contributed equally to this work

This work was partially supported by NCI grant R01 CA104729

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 4395. doi:10.1158/1538-7445.AM2011-4395
Abstract 440: Characterization and comprehensive proteomic analysis of exosomes derived from prostate cancer cell lines
Authors: Hosseini-Beheshti Elham , Pham Steven , Adomat Hans , (Tomlinson) Guns Emma S.
Published: June 2012

Background: Exosomes are extracellular nano-vesicles which are secreted upon fusion of multivesicular bodies with the plasma membrane and present in most body fluids. They have been shown to be derived from a multitude of cell types and, while it is postulated that they are important for membrane trafficking as communication vesicles, their relevance in cancer and specifically prostate tumour growth and progression has yet to be determined. Factors such as stage of cancer, cell type and cell cycle could affect the amount and composition of exosomes formed and secreted. While the mechanisms underlying exosome formation and secretion are not fully understood, characterization of exosomes derived from different androgen dependent and independent prostate cancer cell lines (LNCaP, C4-2, VCaP, DU-145, PC-3) versus normal epithelial prostate cell line (RWPE-1) indicate novel information about their possible role in prostate cancer.

In Addition proteomic analysis of exosomes derived from different prostate cancer cell lines reflects differential protein content within these entities and could be considered for prognostic potential. Other studies on tumour-related microvesicles suggest the role of these selectively enriched exosomes in cancer progression therefore the presence of differential protein markers in and on prostate cancer cell derived exosomes may yield potential biomarkers for cancer diagnosis and therapeutic response. In this study we characterized exosomes derived from different prostate cancer cells and explain the role of specific proteins present within exosomes which are involved in prostate cancer progression.

Methods and Results: Exosomes were purified from the conditioned media(CM) from a panel of five prostate cancer cells and ‘normal’ RWPE-1 cells after 72 hours serum free media treatment. Using differential centrifugation cell debris and protein aggregates were removed from CM. Finally exosomes were isolated in a 30% sucrose cushion using ultracentrifugation. Further analysis using transmission electron microscopy detected the integrity of purified exosomes. Western blot analysis was also used to probe for different exosome markers including Actin, HSP70, HSP90, Rab5, CD9 and CD63. Our proteomic data shows that exosomes derived from specific prostate cancer cells present potential markers of prostate cancer progression.

Conclusion: This study highlights a potential for differential protein composition of exosomes as a source of diagnostic biomarkers in addition to therapeutic targets for prostate cancer via non-invasive testing.

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 440. doi:10.1158/1538-7445.AM2011-440
Abstract 4424: Antitumor activities of targeted therapeutic agents in preclinical models of gastric cancer
Authors: Fan Conglin , Pascual Bernadette , Wu Ying , Whalen Pamela , Wang Yuli , Pocalyko David , Li Gang
Published: June 2012

Gastric cancer (GC) is one of the most prevalent cancer types in Asia, and the 2nd most common cause of cancer deaths worldwide. Although considerable advances have been made with the use of improved surgical techniques and chemotherapy regimens, the prognosis for most GC patients remains poor. Therefore there is an urgent medical need for novel, effective targeted therapeutic agents for GC. One of the key drivers for a successful drug discovery program is utilization of predictive preclinical models that represent the patients of interest. Through internal efforts and collaborations we have developed subcutaneous (SC), orthotopic, syngeneic and patient-derived xenograft (PDX) models for target validation and pharmacological studies. Here we report the establishment of various cell line-derived mouse models of GC, and their validation with targeted therapeutic agents. In SC models of MKN-74, NCI-N87 and MKN-45, sunitinib malate significantly inhibited tumor growth. To establish orthotopic GC model, luciferase-expressing MKN-74 and NCI-N87 cells were first SC implanted in athymic nude mice. Four weeks after in vivo propagation, tumor fragments (1 mm3) were surgically implanted to the stomach wall in the greater curvature region. Primary tumor growth and spontaneous metastasis were monitored by bioluminescent imaging. Sunitinib malate treatment inhibited primary tumor growth but the effect on metastasis was inconclusive. Furthermore, PF-299804, an orally available irreversible pan-HER tyrosine kinase inhibitor, inhibited in vivo growth of NCI-N87 (with ERBB2 amplification), but not of AGS (without ERBB2 amplification). Both sunitinib malate and PF-299804 were well tolerated by the animals during the course of the studies. These results support anti-angiogenesis and as a valid approach for effective treatment of GC, and preclinically demonstrated the importance of patient selection for targeted agents.

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 4424. doi:10.1158/1538-7445.AM2011-4424