bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4855: Use of blocker locked nucleic acid (LNA™) oligonucleotides in cycle sequencing for improved limits of detection (BLOC-Sequencing)
Authors: Legendre Benjamin , Wu Grant , Stoddard Jason , Scott Jaclyn , Johnson Emily , Sommer Julie , Murphy Tammy , Echtenkamp Karissa , Richardson Katherine
Published: June 2012

Epidermal growth factor receptor (EGFR) antagonists, such as cetuximab and panitumumab, are therapeutic agents that can be effective in colorectal cancer (CRC) treatment. It has been shown that 40% of CRC tumors have activating K-RAS exon 2 codon 12 and 13 mutations and that these mutations may be associated with a poor response to EGFR antagonists. Very high sensitivity detection of such diagnostic biomarkers is necessary to determine the presence or emergence of drug resistant tumor cell populations.

The use of Locked Nucleic Acid (LNA™) containing oligonucleotides (oligos) have been used in microRNA (sample preparation), RNA (in situ hybridization), and DNA (SNP detection using allele specific PCR) applications. The advantage of using LNA-containing oligos is that the denaturation temperature of LNA-oligo:DNA duplex is increased as compared to the DNA:DNA duplex

To further the limit of detection of Sanger sequencing, a LNA-based approach has been developed to selectively block the sequencing of the wild-type DNA and allow the sequencing of DNA containing any mutation (BLOC-Sequencing). During cycle sequencing, an additional annealing step is added to hybridize the LNA containing oligo to the template DNA. Then, a denaturing step is performed at a temperature at which the LNA-containing oligo remains annealed to the wild-type sequence; however, the LNA oligo denatures from the mutant sequence. The sequencing primer then anneals to the mutant sequence and subsequently extended. Since LNA-containing oligos block amplification of the wild-type sequence, the mutant-containing DNA will be sequenced in a non-biased manner.

Preliminary results using two sequences in a 1:1 ratio differing in only 2 nucleotides within the range where the LNA blocking oligo anneals demonstrated complete blocking of one sequence (sequence 1) where the LNA containing oligo was 100% complementary. The unblocked sequence (sequence 2) sequenced successfully, with minimal background due to sequence 1. Straight Sanger sequencing results of the 1:1 mixture without the LNA blocking oligo gave equal proportion of the two sequences present.

To show applicability for use of this methodology in CRC samples, the limit of detection for multiple codon 12 and codon 13 K-RAS exon 2 mutations with and without the addition of the K-RAS exon 2 wild type specific LNA oligo in both the forward and reverse directions will be demonstrated. In addition, we will compare the results of 30 previously genotyped FFPE samples with this technique.

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 4855. doi:10.1158/1538-7445.AM2011-4855
Abstract 4870: High-throughput proteomics using stable isotope labelling with amino acids in cell culture (SILAC) reveals potential modulators of androgen-independent prostate cancers
Authors: Saraon Punit , Batruch Ihor , Smith Chris , Mizokami Atsushi , Jarvi Keith , Diamandis Eleftherios P.
Published: June 2012

Prostate cancer is the most common malignancy and the second leading cause of cancer related deaths in men. One common treatment is androgen-deprivation therapy, which reduces symptoms in most patients. However, over time patients develop tumours that are androgen-independent and ultimately fatal. The mechanisms that cause this transition remain largely unknown, and as a result, there are no effective treatments against androgen-independent prostate cancer. As a model for androgen-independence, we used the LNCaP cell line which is naturally androgen-dependent grown in androgen depleted conditions for a 6 month period to generate the androgen-independent cell line LNCaP-SF. Preliminary experiments showed that the LNCaP-SF cell line had higher proliferation/viability rates in androgen-depleted conditions, and had increased expression of the androgen receptor protein, a key modulator in prostate cancer progression. Our next goal was to assess to differential global protein expression as well as differential expression of secreted proteins from each of the two cell lines. To identify differentially expressed proteins, we utilized Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) coupled to mass spectrometry. LNCaP cells were labelled in growth media containing light-Arg/Lys whereas LNCaP-SF was labelled in heavy Arg-Lys media. After proteomic analysis, we were able to quantify 3355 proteins with at least one peptide hit. From these proteins, using stringent criteria, 99 and 82 proteins were found to be up-regulated and down-regulated respectively, in LNCaP-SF cells compared to LNCaP cells. Using various bioinformatics tool, we found that genes involved in fatty acid degradation and ketogenesis as important pathways that with over-expressed in LNCaP-SF cells. We performed qPCR validation of genes involved in each of these pathways and found that LNCaP-SF cells generally had an increase in the expression of many these genes, compared to LNCaP cells. These preliminary results show that genes involved in fatty acid degradation and ketogenesis pathways could serve as potential biomarkers for androgen-independent prostate 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 4870. doi:10.1158/1538-7445.AM2011-4870
Abstract 4878: Application of a novel nano-immunoassay platform to assess changes in cIAP1 in response to the SMAC-mimetic, LCL161
Authors: Yeleswarapu Lakshmi , Nuti Shanthy , Graber Armin , Gardner Humphrey , Barrett Carl , Ramkumar Thiruvamoor
Published: June 2012

Introduction: cIAP1 is member of a class of inhibitor of apoptosis proteins (IAP) that function in a regulatory role to prevent unintended cell death by apoptosis. LCL161 is a small molecule antagonist of IAPs that reverses cIAP1 inhibition of caspases, causing apoptosis. The present study explores the use of the Nano Pro 1000, a novel proteomic platform to measure the changes in cellular cIAP1 in response to treatment with LCL161. Methods: SKOV3 cells, peripheral blood mononuclear cells (PBMC) and hair follicles collected from healthy donors were treated with LCL161 for different times and subjected to capillary iso-electric focusing. Immunodetection was performed using antibodies directed against cIAP1 and the signal was quantified using HRP-labeled chemiluminescence reagents. Data from the Nano Pro were compared to results from western blots run in parallel. Results: We report that results from the nano-immunoassay are reproducible and correlate well with the western blotting data. Modulation in the cellular levels of cIAP1 in response to LCL161 treatment could be captured in samples containing as little as 1 ng of total protein. Similar modulation of cIAP1 was seen upon exposure to LCL161 in the studied test matrices. Conclusions: This novel proteomic tool is more sensitive, specific and quantitative compared to the currently available option of western blotting assays which are labor intensive, and have a relatively low throughput. Measurable signals corresponding to cIAP1 levels can be detected in matrices like PBMCs and hair follicles. The modulation of cIAP1 levels in these matrices mirror that seen in SKOV3 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 4878. doi:10.1158/1538-7445.AM2011-4878
Abstract 492: ALDH1A1 expression predicts for a tumorigenic subset in primary cultures of malignant pleural effusions (MPE)
Authors: Basak Saroj Kumar , Mysore Veena , Lai Chi , Oh Scott , Srivatsan Eri , Batra Raj
Published: June 2012

Aldehyde dehydrogenases (ALDHs) are a group of enzymes that are responsible for oxidation of a variety of aldehydes. ALDH isoforms (ALDH1A1 and ALDH3A1) are highly expressed in non-small cell lung cancer (NSCLC) cell lines. Recent evidence suggests that elevated levels of these enzymes may be associated with malignant behavioral properties of cell subsets in epithelial malignancies. It is not known whether cell subsets with high ALDH expression are present in advance stage lung cancer patients, or whether ALDH expression is associated with malignant behaviors (e.g. tumorigenicity) in lung cancer cell subsets. We hypothesized that the ALDH1A1-biomarker is present in advanced lung cancer, and that as in other epithelial malignancies, its expression predicts for lethal behavioral properties.

We isolated lung tumor cells from advanced stage lung cancer patients (MPE), and maintained them in primary culture in presence of MPE fluid (an “autologous tumor microenvironment”). ALDH labeling by immunohistological methods revealed high intratumoral variability with respect to biomarker expression. To live detect MPE-primary culture cells, the Aldefluor (StemCell Technologies Inc.) method of Fluorescence Activated Cell Sorting (FACS) was used. 4-8% of MPE-primary culture cells expressed high level of ALDH1A1, whereas the majority of the cultured cells expressed intermediate or low levels of ALDH1A1. Interestingly, the ALDH1A1hi cell subset also expressed high levels of the CD44 surface biomarker. CD44 is another widely studied biomarker that has been associated with “cancer stem cell” properties. The ALDH1A1hi subset co-expresses 2 to 20 times higher CD44 expression, as compared to the ALDH1A1lo subset. Importantly, the cells that dually label for CD44hi and ALDH1A1hi exhibit increased soft agar colony forming efficiency in vitro, and increased tumorigencity in vivo. Limiting dilution studies of xenotransplantation in NOD/SCID mice also provide experimental evidence that clinical (MPE) biospecimens of some lung cancers possess ALDH1A1hi/CD44hi “cancer stem cells” (as evidenced by tumorigenesis following less than 1000 implanted cells). The tumors that emerge from the implantation of these lung “cancer stem cells” display phenotypic heterogeneity with respect to both CD44 and ALDHA1 expression. Collectively, these data provide experimental evidence for tumorigenic subsets in clinical lung cancer biospecimens, and suggest that ALDHA1 and CD44 expression are jointly regulated by a common oncogenic program in lung 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 492. doi:10.1158/1538-7445.AM2011-492
Abstract 4938: Extracellular miRNAs have potential as minimally-invasive biomarkers for predicting response to HER2-targeted agents, including Trastuzumab, as used in breast cancer
Authors: Corcoran Claire , Rani Sweta , Browne Brigid , Crown John , O'Donovan Norma , O'Driscoll Lorraine
Published: June 2012

The identification of minimally-invasive biomarkers, in efforts to aid in patient-tailored disease management, has become a key focus of cancer research. Recent studies by ourselves and others have reported the existence of circulating miRNAs associated with breast cancer. Despite this progress, resistance to anti-cancer treatment remains a major obstacle in the treatment of this disease and unfortunately there are no reliable methods of predicting sensitivity/resistance to anti-cancer agents.

This study aimed both to (i) identify miRNAs differentially-expressed in the breast cancer cell line SKBR3 and its Trastuzumab-resistant variant, SKBR3-H, developed by exposure to Trastuzumab for 6 months; and to (ii) investigate if corresponding differential levels of miRNAs were detectable in conditioned media (CM) from these cells, reflecting the relative sensitive/resistance profile of the SKBR3-H comparing to age-matched control SKBR3 cells.

Global analysis of miRNAs was successfully performed on biological triplicate RNA specimens isolated from SKBR3 cells, SKBR3-H cells and on their respective CM, using TaqMan low density arrays representing 667 human miRNAs. 71.5% (477/667) miRNAs were detected in SKBR3; most (85%; 406/477) of which were detected in its CM. For SKBR3-H, 75.7% (505/667) miRNA analyzed were present in the cells, with 70% (354/505) of these also detected in corresponding CM. Of the 187 miRNAs up-regulated by at least 2-fold in SKBR3-H compared to SKBR3 cells, 6.42% (12/187) were up-regulated in corresponding CM. Of the 62 miRNAs down-regulated by at least 2 fold in SKBR3-H compared to SKBR3 cells, more than half (53.2%; 33/62) were down-regulated in corresponding CM. Several differentially regulated miRNAs were selected from TLDA results and subsequently validated in triplicate by qRT-PCR.

Overall, the many hundred (∼500) miRNAs detected in CM further supports the existence of extracellular miRNAs and so their potential as minimally-invasive biomarkers. Furthermore, we identified a number of miRNAs that may help predict response to Trastuzumab. Our current research involves investigating the relative expression of miRNAs in SKBR3 variants resistant to other HER2-targeted agents and also examining the potential target genes for miRNAs identified as of most relevance here. Exploring the occurrence of these miRNAs in relevant patients’ serum specimens is now warranted to investigate clinical application.

Acknowledgements: Science Foundation Ireland's SRC award to MTCI (08/SRC/B1410).

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 4938. doi:10.1158/1538-7445.AM2011-4938
Abstract 4950: miR-326 is critical for VEGF-C induced CTTN expression and metastasis of esophageal squamous cell carcinoma
Authors: Chiou Jean , Lin Chih-Chun , Jan Yi-Hua , Hsiao Michael , Su Jen-Liang
Published: June 2012

Esophageal cancer is a major cause of cancer deaths in most Asian countries, which is so called Asian esophageal cancer belt. Recent studies have demonstrated that, Cortactin (CTTN), a cytoskeletal protein frequently overexpressed in many types of human cancers including esophageal squamous cell carcinomas. Animal studies suggest that CTTN overexpression increases esophagus tumor aggressiveness, possibly through promotion of tumor invasion and metastasis. Vascular endothelial growth factor (VEGF)-C, ligand for the VEGF receptor-3, is critical for cancer metastasis through induces the formation of tumor-associated lymphatic vessels and facilitate the mobility of tumor cells. However, the detail molecular mechanisms involved in VEGF-C/VEGFR-3 axis-mediated tumor progression is not clear yet. Besides, the biological significance of the correlation between VEGF-C and CTTN in cancer cells is completely unknown. In this study, we found CTTN is critical for VEGF-C-mediated migration and invasion of esophageal squamous cell carcinoma (ESCC) cells. We also found that miR-326, which could target the 3’UTR of CTTN, is involved in VEGF-C-induced CTTN expression and subsequent migration and invasion of ESCC cells through an Src-dependent pathway. Taken together, our results show that VEGF-C up-regulates CTTN protein expression, resulting in enhanced cell invasion, tumorgenesis and metastasis through Src-mediated down-regulation the expression of miR-326.

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 4950. doi:10.1158/1538-7445.AM2011-4950
Abstract 4954: Comparison of miR expression patterns in plasma, serum, and cell line cMV
Authors: Pawlowski Traci L. , Yeatts Kimberly , Millis Meredith , Radebach Katrina , Kojima Yuka , Zhong Jason , Stark Adam , Kuslich Christine
Published: June 2012

Circulating microvesicles (cMV) isolated from either blood plasma or serum can yield genetic material sufficient for informative microRNA (miR) expression analysis. The ability to purify RNA from cMV isolated from several sources was compared, and the miR expression patterns were determined. cMV were isolated from the blood plasma and serum of men with and without prostate cancer, as well as from healthy and prostate tumor tissue and prostate cancer cell lines. Total RNA was extracted from the plasma and cell line cMV using the Qiagen miReasy kit and from the serum cMV using the ExoMir extraction method. The miR expression was determined for each source using Agilent V3 miRNA microarrays. Each sample (300 ng) was hybridized to the microarrays, and the acquired data were analyzed with the GeneSpring software package. Expression patterns for miR from cMV isolated from the plasma and serum of men with and without prostate cancer were evaluated for significant differences. Hierarchical clustering on both samples and genes demonstrated a distinct expression pattern for miR from plasma cMV compared with miR from cell line and tumor tissue cMV. These findings demonstrate that cMV isolated from the peripheral blood offer a unique and potentially clinically relevant source of miR for blood-based 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 4954. doi:10.1158/1538-7445.AM2011-4954
Abstract 499: High-level expression of the stem-cell marker CD133 in clear cell renal cell carcinoma with favorable prognosis
Authors: Kim Kyungeun , Ihm Hyojin , Ro Jae Y , Kang Sunghee , Kang Munjung , Cho Yong Mee
Published: June 2012

The cancer stem cell (CSC) model suggests that high levels of CSCs within a tumor are associated with poor prognosis. This study aims to investigate the expression of the stem cell marker CD133 in clear cell renal cell carcinoma (ccRCC) and its prognostic significance. The expression of CD133 was examined in 140 cases of ccRCC using immunohistochemistry. Ki-67 and Oct-4 were double immunostained with CD133 to evaluate the proliferative activity and the stemness of CD133-expressing cells, respectively. We observed CD133 expression in 45 cases (32.1%) and high levels of expression were associated with a macro-/microcystic pattern, no sarcomatoid changes, and non-metastatic disease. The Ki-67 labeling index tended to be lower in CD133-expressing ccRCCs compared with non-expressing tumors. CD133-expressing tumor cells rarely expressed Oct-4. CD133 was also highly expressed in renal intratubular neoplasia. This study demonstrated CD133 expression in pre-malignant and malignant renal diseases. We observed a high degree of CD133 expression in ccRCC with more differentiated morphology and non-metastatic disease, suggesting CD133 as a favorable prognostic marker. These results also imply that CD133 as a single marker may not be sufficient for CSC identification in ccRCC, and therefore, more specific CSC markers should be developed.

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 499. doi:10.1158/1538-7445.AM2011-499
Abstract 4996: The VEGF quadruplex-forming sequence inhibits lung cancer cell growth
Authors: Muench David , Thomas Shelia D. , Miller Donald M. , Sedoris Kara C.
Published: June 2012

Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGF q), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with an oligonucleotide encoding VEGF q inhibits cell growth by decreasing VEGF expression and subsequent signaling through VEGF receptor 2 (FLK-1). To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGF q or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Our results demonstrate that VEGF q formed a parallel quadruplex structure and showed remarkable serum and intracellular stability. Confocal and flow cytometry analysis of cells treated with FITC-labeled VEGF q showed prominent uptake and nuclear/cytoplasmic localization in contrast to MutVEGF. Treatment of A549 cells with VEGF q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM) as determined by MTT assay, which was not a result of cell cycle arrest. Matrigel analysis also showed a significant reduction in tumor invasiveness after 24 h with VEGF q treatment. Although uptake of VEGF q occurred in non-transformed Hs27 human fibroblast cells, no change in cell growth was evident, indicating that the anti-proliferative effects of VEGF q are specific for malignant cells. Inhibition of cell proliferation and invasion corresponded with decreased VEGF protein expression resulting in decreased FLK-1 signaling and phosphorylation of the target proteins ERK 1/2 and AKT. These results demonstrate striking growth inhibition and decreased invasion of A549 cells in response to VEGF q related to inhibition of VEGF expression and signaling through FLK-1 and have significant therapeutic implications for the treatment of non-small cell 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 4996. doi:10.1158/1538-7445.AM2011-4996
Abstract 5000: Tolerogenic transcription regulator Foxp3 controls the expression of immunosuppressive cytokines in melanoma cells
Authors: Olson Samuel Y. , Chen Cynthia , Marquez Diana C. , Bruhn Kevin W. , Craft Noah A. , Garban Hermes J.
Published: June 2012

Foxp3 is a critical transcription regulator that is found commonly expressed in a subset of immunosuppressive CD4+/CD25+ T cells characterized as regulatory T cells (Tregs) in the immune system. Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators exhibiting both, activation and repression of transcription. Foxp3 is critically important for the development and function of Tregs and the consequent definition of immune-tolerance. Ectopic Foxp3 expression has been shown to -phenotypically and functionally- convert T cells to Tregs. Recently, we have characterized the expression of Foxp3 in a variety of human and mouse cancerous cells, either in tissue or in cultured cell lines. Although of cardinal importance, the specific role and mechanisms involved in the Foxp3-mediated immunosuppressive activity remains elusive. Thus, we hypothesize that tumor-derived Foxp3 is controlling the transcriptional expression of immunosuppressive cytokines by tumor cells and inducing immunotolerance in the tumor microenvironment. We have tested this hypothesis by interrogating the well-characterized B16-C57BL/6 syngeneic mouse model of melanoma. Foxp3-expressing B16-F0 cells (B16-Foxp3+) where specifically and stably knockdown for Foxp3 expression by lentivirus-mediated shRNA (B16-Foxp3-). In order to determine the specific role and mechanisms of melanoma-derived Foxp3 in controlling the expression of immunosuppressive cytokines generated by melanoma cells, we have evaluated the differential cytokine expression profile between the B16Foxp3+ and the B16-Foxp3- cultured cells using Multiplex Bead Immunoassay, Immunoblotting Assay of Proteins and Semiquantitative RT-PCR. Our results demonstrated that the suppression of expression of Foxp3 in melanoma cells showed a significant downregulation of a selected group of immunosuppressive cytokines, in particular IL-4, IL-6, IL-10, IL-12, TNF-alpha and GM-CSF. In contrast, Foxp3 did not have an appreciable effect on the expression of IL-1, IL-2, IL-5, and IFN-gamma. Altogether, our data suggest the specific role of tumor-derived expression of FOXP3 in the control of the expression of tolerogenic/immunosuppressive cytokines secreted by melanoma cells to the tumor microenvironment. Furthermore, selective suppression of Foxp3 expression and/or activity in cancerous cells may help to develop better and more effective targeted immunotherapy as treatment for malignant melanoma.

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 5000. doi:10.1158/1538-7445.AM2011-5000
Abstract 5008: Epidermal growth factor downregulates expression of neutrophil gelatinase-associated lipocalin (NGAL) through E-cadherin in pancreatic cancer cells
Authors: Tong Zhimin , Chakraborty Subhankar , Sung Bokyung , Kaur Sukhwinder , Aggarwal Bharat B. , Mani Sendurai , Bresalier Robert S. , Batra Surinder , Guha Sushovan
Published: June 2012

Background: Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, is a secreted protein that is upregulated in multiple cancer types. Our group previously reported that NGAL overexpression significantly blocked invasion and angiogenesis in pancreatic ductal adenocarcinoma (PDAC). We and others have also demonstrated loss NGAL expression in advanced stages of PDAC. However, little is known about mechanisms that regulate NGAL expression in PDAC. As EGF-EGFR axis is significantly upregulated in PDAC, we examined EGF-mediated NGAL regulation in these cells.

Methods: NGAL-positive AsPC-1 and BxPC-3 cells were used as model system. Quantitative RT-PCR, western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or shRNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay (EMSA).

Results: NGAL expression was positively associated with tumor differentiation and was significantly downregulated after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin downregulation was partly through the EGF receptor (EGFR)-dependent MEK1/2-ERK1/2 signaling pathway, which was demonstrated using pharmacologic inhibitors of EGFR (AG1478) and MEK1/2 (U0126 and PD98059). In addition, E-cadherin downregulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued inhibition of NGAL expression by EGF. Furthermore, EGF in part through E-cadherin reduced NGAL promoter activity by blocking NF-κB activation.

Conclusion: We demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect is partly mediated through activation of the EGFR-MEK1/2-ERK1/2 signaling pathway, which in turn downregulated E-cadherin with a subsequent reduction in NF-κB activation. Our findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC.

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 5008. doi:10.1158/1538-7445.AM2011-5008
Abstract 5056: HuGAL-FR21, a humanized monoclonal antibody to Fibroblast Growth Factor Receptor 2, effectively inhibits the growth of gastric tumor xenografts
Authors: Park Hangil , Wang Lihong , Chhim Sophea , Ding Yi , Kim Jin
Published: June 2012

The family of 22 fibroblast growth factors include potent mitogenic and angiogenic factors believed to play a role in the growth of many tumors. The various FGFs bind to only four tyrosine kinase signaling receptors, Fibroblast Growth Factor Receptors 1-4 (FGFR1-4). FGFR2 is a major cellular receptor for FGF2 (basic FGF), FGF7 (keratinocyte growth factor, KGF) and FGF10; and mutation or overexpression of FGFR2 is associated with a number of cancers, notably endometrial cancer and gastric carcinoma. In this study, we explored the therapeutic potential of the HuGAL-FR21 humanized monoclonal antibody (mAb) derived from our blocking anti-FGFR2 mAb, GAL-FR21.

We previously showed that GAL-FR21 binds to only the FGFR2IIIb isoform of FGFR2, which is expressed on epithelial tissues and thus more commonly on carcinomas. Using well-established humanization techniques, GAL-FR21 was humanized to create HuGAL-FR21, which binds human FGFR2IIIb with at least as high affinity as GAL-FR21. HuGAL-FR21, like GAL-FR21, also bound well to monkey and mouse FGFR2IIIb, facilitating study of this mAb in animal models. In functional assays, HuGAL-FR21 inhibited binding of FGF2, FGF7 and FGF10 to FGFR2IIIb as completely as GAL-FR21. And HuGAL-FR21 inhibited FGF7 or FGF10-induced proliferation of the Ba/F3 cells transfected with FGFR2IIIb, actually better than GAL-FR21. Finally, HuGAL-FR21 inhibited phosphorylation of cellular FGFR2 induced by FGF7 and FGF10 as well as GAL-FR21.

There are two major types of gastric cancer, well-differentiated and poorly differentiated, and published results show that many poorly differentiated tumors overexpress FGFR2, which is in fact the oncogenic driver for these tumors. We therefore tested the ability of HuGAL-FR21 to inhibit growth of xenografts of two cell lines, SNU-16 and OCUM-2M, derived from FGFR2-overexpressing, poorly differentiated gastric tumors. HuGAL-FR21, administered i.p. at 2.5 or 5 mg/kg twice per week, strongly inhibited the growth of established xenografts of both these cell lines. We conclude that FGFR2 is a promising molecular target, and that HuGAL-FR21 is a viable clinical candidate for the treatment of gastric and possibly other cancers.

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 5056. doi:10.1158/1538-7445.AM2011-5056
Abstract 5071: Application of a novel protein biomarker discovery platform to identify biomarkers for improved diagnosis of prostate cancer
Authors: McAndrew Michael , Wheeler Colin , Koopmann Jens , Uddin Ezam , Agashe Pooja , Workman Nick , Anson John
Published: June 2012

The issues surrounding the use of prostate-specific antigen (PSA) in the diagnosis of prostate cancer (PCa) are well documented and the need for a molecular diagnostic test with greater discriminatory power is clear. The development of autoantibodies associated with prostate cancer has also been described. In general, the appearance of such antibodies can precede disease symptoms by many years, making them attractive as potential biomarkers for early diagnosis.

We have developed a unique “functional protein” array platform which utilises correctly folded proteins and has the ability to display native, discontinuous epitopes. The reproducibility of the platform is exceptionally good, making it possible to screen statistically meaningful numbers of samples. Following on from a successful pilot study, where panels of autoantibody biomarkers exhibiting a specificity and sensitivity for PCa superior to PSA were identified using the platform1, we have validated this approach in a large-scale analytical study.

The current analytical study involving approximately 1800 samples was primarily designed to identify panels of biomarkers with the ability to distinguish between PCa (n=400) and control samples from patients with benign prostatic hypertrophy (BPH, n=406). BPH can present with similar symptoms to PCa and can also result in elevated PSA levels. Additional sample cohorts included prostatitis, other cancers of various origin and non-disease/healthy controls (n=400). All samples were age, ethnicity and gender matched.

The products of 1296 unique genes (1330 proteins), chosen for their association with disease, signal transduction and cancer autoimmunity, are immobilized on each array. Serum samples (n=1781) were analysed using the arrays as described previously1. Data were split into test and training sets and analyzed with several classification algorithms to identify classifiers which would successfully distinguish case from control samples. Data were repeatedly split into test and training sets and analysis cycles repeated until a stable set of classifiers was identified. At the time of writing, data is still under analysis; however, early indications are encouraging and suggest that panels of biomarkers with a performance that significantly exceeds that of PSA may be identified. Full results of the study will be presented.

1 McAndrew et al Development of a panel of biomarkers for the diagnosis of prostate cancer (2010) Molecular Diagnostics in Cancer Therapeutic Development conference

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 5071. doi:10.1158/1538-7445.AM2011-5071
Abstract 5093: The molecular genetics of radiogenic breast cancer
Authors: Wade Mark A. , Batey Michael A. , Rand Vikki , Russell Lisa J. , Bacon Chris M. , Allan James M. , May Felicity E.B
Published: June 2012

The aim of this study is to identify the underlying molecular genetic mechanisms of radiation induced breast carcinogenesis. Studies have shown that women exposed to ionising radiation at a young age are more at risk of developing breast cancer than older women exposed to the same level of radiation. Higher levels of estrogen are present in young women and estrogen has a known transforming effect on breast epithelial cells. One hypothesis suggests that radiation and estrogen synergise to drive breast epithelial cell transformation. We have developed an in vitro model of radiation-induced breast epithelial cell transformation in order to investigate genetic alterations associated with breast cell transformation.

The immortalised, non-transformed breast epithelial cell line MCF-10A was exposed to fractionated doses of X-rays in the presence or absence of exogenous estrogen. We have shown that radiation treated cells display evidence of transformation, including loss of contact inhibition, increased cell invasion, disrupted acini formation and tumor formation in immunocompromised Rag2−/− γ−/− mice. Analysis of radiation treated cells by SNP array karyotyping identified focal gene deletion (EP300 and OCT-1) and amplification (c-MYC) which may be linked to radiation-induced breast cell transformation. Gene deletion and amplification was confirmed by Fluorescent In Situ Hybridization analysis and alterations in expression have been confirmed by western analysis.

EP300 is a transcriptional co-activator and a putative tumor suppressor. Somatic mutations in EP300 have been found in solid tumours and the gene is located in a region of chromosome 22 affected by loss of heterozygosity in numerous cancers. Oct-1 encodes an octamer binding transcription factor that has been associated with regulating DNA damage response through interactions with BRCA1 and GADD45. c-MYC is a known proto-oncogene that encodes a transcription factor involved in cell proliferation, cell cycle regulation and apoptosis.

Analysis of our in vitro model of radiation-induced breast epithelial cell transformation has identified genetic alterations that have established roles in mediating cellular response to DNA damage and which might be key events in the development of radiogenic 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 5093. doi:10.1158/1538-7445.AM2011-5093
Abstract 5102: Increased plasma levels of MAPRE1, an APC interacting protein, preceding a diagnosis of colorectal cancer
Authors: Ladd Jon , Buson Tina B. , Johnson Melissa M. , Zhang Qing , Pitteri Sharon J. , Wang Hong , Prentice Ross L. , Hanash Samir
Published: June 2012

Longitudinal blood collections from cohort studies provide the means to search for proteins associated with disease processes prior to diagnosis. We have investigated pre-diagnostic plasma samples to determine quantitative differences in plasma protein between subjects subsequently diagnosed with colorectal cancer (CRC) and matched controls that remain cancer free during an 18 month period of active follow-up. In-depth quantitative proteomic profiling was performed on 90 pre-diagnostic plasma samples. Experiments compared nine pools each consisting of plasma aliquots from 10 CRC subjects diagnosed within 18 months after blood draw and 10 matched controls. MAPRE1 was confirmed in a set of 58 pairs of plasma from newly diagnosed CRC cases and controls using enzyme linked immunosorbent assay (ELISA). MAPRE1 was further tested by ELISA in a second independent set of 100 pre-diagnostic CRC plasma samples diagnosed within 18 months after blood draw and 100 matched controls. Proteomic analysis of pre-diagnostic plasma samples collected prior to diagnosis of CRC yielded a set of proteins with elevated concentrations (p <0.05) in cases compared to control that included MAPRE1 which is a binding partner for APC. MAPRE1 and carcinoembryonic antigen (CEA) were assayed by ELISA in newly diagnosed CRC samples, yielding equally significant elevation (p <0.05) among cases relative to controls for each protein with areas under the curve (AUCs) of 0.778 and 0.782, respectively. Further testing in an independent set of pre-diagnostic samples confirmed predictive value of MAPRE1 by receiver operator characteristic (ROC) achieving an AUC of 0.675 within 7 months of diagnosis. This is the first reported evidence of elevated circulating levels of MAPRE1 in colorectal cancer patient plasma. Plasma levels increased with development of CRC. Because of MAPRE1's interaction with adenomatous polyposis coli protein (APC), a known target of mutation in a majority of CRC, increased circulating levels of MAPRE1 may be an early indicator of CRC 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 5102. doi:10.1158/1538-7445.AM2011-5102
Abstract 5114: Mass spectrometric immunoassays for quantitative determination of protein biomarker variants
Authors: Nedelkov Dobrin , Trenchevska Olgica , Pupinoska Aleksandra , Phillips David , Kamcheva Elena , Romevska Slobodanka , Kiernan Urban A.
Published: June 2012

Post-translational modifications and genetic variations give rise to protein variants that significantly increase human proteome complexity. Modified proteins also play an important role in biological processes. Mass spectrometry, coupled to immunoaffinity separations, provides an efficient mean for simultaneous detection and quantification of protein variants. In Mass Spectrometric Immunoassays, affinity interactions are utilized to selectively retrieve specific proteins from complex biological fluids, and are followed by mass spectrometric analysis of the targeted proteins. These assays are similar to traditional enzyme immunoassays in that they use antibodies as reagents for affinity retrieval of specific proteins, but instead of enzymatic reaction for indirect protein detection, mass spectrometric analysis is used for direct identification of the proteins and their modifications.

Described here are mass spectrometric immunoassays for targeted quantitative proteomics analysis of specific protein modifications. Assays for several protein biomarkers exhibiting posttranslational modifications were developed, including beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin. The following assay performance characteristics were examined (and determined): LOQs (sufficient for quantification of normal/healthy levels of the proteins from human serum or plasma); intra- and inter-assay precision (CVs of <10%), linearity and spiking recovery (90-110% range); specificity (<5% cross reactivity with other proteins), and correlation with ELISA-generated results (acceptable Passing & Bablok fits and Cusum linearity p-values of >0.1). The new assays were then utilized to determine the individual concentration of the specific protein variants across larger cohorts of samples (n=500), demonstrating the ability to fully quantify individual forms of post-translationally modified proteins.

The mass spectrometric immunoassays are ideally suited for detection and quantification of novel protein variants, not known a priori. These MS-based assays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity.

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 5114. doi:10.1158/1538-7445.AM2011-5114
Abstract 5120: Large-scale proteome analysis reveals complex molecular abnormalities in gastric cancer
Authors: Guo Tiannan , Zhu Yi , Ng Wai Har , Kon Oi Lian , Sze Siu Kwan
Published: June 2012

Cancer proteomes have not been comprehensively studied on a genome-wide scale. In this work, we have catalogued the proteome of a human gastric cancer cell line, SNU5, by integrative analysis of multiple proteomic data sets generated from mitochondria, nucleus and membranes, in addition to the phosphoproteome and whole cell proteome. We report a total of 10440 proteins, of which 8578 were identified with at least two unique spectra and 7042 with more than one unique peptide. We localized gastric cancer proteins to specific organelles at higher resolution than in current knowledgebases. Proteins in sub-organelles such as the Cajal body were identified. Bioinformatic analysis suggests that the proteome map can be a tool of disease taxonomy. Seventeen oncogenesis-associated signaling pathways, including mitochondrial dysfunction, HGF signaling pathway and Eph receptor pathway, were over-represented in the gastric cancer proteome. We also documented the expression of more than 100 protein kinases in gastric cancer. Selected proteins were further investigated in primary gastric cancer tissues. Our approach demonstrates that bioinformatic integration of proteomic data sets substantially increases the depth of proteomic studies. This deep proteome map of cancer cells should aid understanding of the systems biology of gastric 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 5120. doi:10.1158/1538-7445.AM2011-5120
Abstract 5127: G6-mediated inhibition of Jak2 tyrosine kinase correlates with the cleavage and cellular reorganization of vimentin
Authors: Majumder Anurima , Kirabo Annet , Karrupiah Kanchana , Tsuda Shigeharu , Caldwell-Busby Jennifer , Cardounel Arturo J. , Sayeski Peter P.
Published: June 2012

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including various leukemias, lymphomas, myelomas and the myeloproliferative neoplasms. Using structure-based virtual screening, our lab previously identified a novel Jak2 inhibitor named G6. We showed that G6 has a specific inhibitory effect on Jak2 tyrosine kinase activity and potently suppresses Jak2-mediated cellular proliferation. To elucidate the biochemical and cellular mechanisms by which G6 inhibits Jak2-mediated cell proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 hours with either vehicle control (DMSO) or 25 µM of the drug and then compared their protein expression profiles using two-dimensional gel electrophoresis. One protein that was found to be differentially expressed was the intermediate filament protein vimentin. Specifically, we found that full length vimentin was robustly expressed in untreated cells and absent in G6 treated cells. We hypothesized that inhibition of Jak2-V617F dependent pathogenic cell growth by G6 correlates with the cleavage and cellular reorganization of intermediate vimentin filaments. To this end, we found that the treatment of HEL cells with G6 induced a time- and dose-dependent cleavage of vimentin. This G6-induced cleavage of vimentin was both Jak2-dependent and calpain-mediated. Using indirect immunofluorescence, we found that G6 treatment resulted in a marked reorganization of vimentin filaments within cells. Finally, using a mouse model of Jak2-V617F mediated neoplasia, we found that G6 decreased the levels of vimentin protein within the bone marrow of G6 treated mice when compared to animals that received vehicle control. Overall, these results demonstrate that G6-mediated inhibition of Jak2-dependent pathogenic cell growth correlates with the proteolytic cleavage of full length vimentin and the reorganization of intermediate vimentin filaments within cells. As such, we may have identified a biochemical and cellular mechanism whereby G6 inhibits Jak2-V617F mediated cellular proliferation.

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 5127. doi:10.1158/1538-7445.AM2011-5127