bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A39: A newly defined role of clusterin in the formation of exosomes derived from prostate cancer cells
Authors: Hosseini-Beheshti Elham , Zoubeidi Amina , Nip Ka Mun , Gleave Martin E. , Guns Emma S.
Published: January 2011

Introduction: Molecular chaperones, including heat-shock proteins (Hsps), help cells cope with stress-induced protein misfolding and aggregation and play prominent roles in cellular signaling and transcriptional regulatory networks asscociated with cancer progression and treatment resistance. Clusterin (CLU) is a stress-activated cytoprotective chaperone, up-regulated in Castration Resistant Prostate Cancer (CPRC), that confers hormone and chemo-resistance. Targeting inhibition of CLU using OGX-011 enhances treatment-induced cell death therapeutic response as is currently in clinical trials of men with advanced prostate cancer. Recent studies have linked cancer with exosome formation; exosomes are 30–100 nm membrane-bound compartments secreted from normal and tumor cells. The mechanisms underlying exosome formation and secretion are not fully understood. Factors such as stage of cancer, cell type and cell cycle could affect the amount and composition of exosomes formed and secreted. Since CLU has chaperone activity with a potent ability to inhibit amorphous and fibrillar aggregation of many different proteins, we hypothesize that it can influence exosome formation and release from androgen independent PCa cells.

Methods-Results: PC3 cells were seeded and grown in DMEM with 5% FBS until 70% confluency. Media was changed to 5% charcoal strip serum DMEM and finally to a serum free media for 72 hours before exosome isolation. Exosomes were purified from the conditioned media using differential centrifugation after remove cell debris and protein aggregates. Further analysis by transmission electron microscopy detected the integrity of purified exosomes and their concentration. Western blot analysis was also used to probe for different exosome markers including Actin, Tubulin, TSG 101, HSP70 and CD36. In this study, we functionally analyzed the role of Clusterin in relation to exosome formation from PC-3 PCa cells. Clusterin was knocked down in PC3 cells using siRNA, and confirmed with western blot analysis. The content of Clusterin in exosomes after knockdown remains unchanged while, in contrast, cells become Clusterin depleted. Upon Clusterin knockdown in PC3 cells we also observe a dramatic reduction in the amount of exosomes formed.

Conclusion: We have found high levels of CLU in exosomes derived from PCa cells. Our data suggests that CLU may serve a role in exosome formation since knocking it down by siRNA dramatically inhibits the quantity of exosomes formed.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):A39
Abstract A40: The incorporation of PTEN/EGFR (DNA) FISH and quantitative biomarker immunofluorescence identifies a phenotype associated with survival in patients with malignant glioma
Authors: Donovan Michael Joseph , Erill Nadina , Puig Pere , Colomer Anna , Boluda Susana , Ferrer Isidre
Published: January 2011

Introduction: Glioblastoma multiforme (GBM) is the most common and malignant of adult glial tumors. Despite best standard of care including surgical resection, radiation and chemotherapy (e.g. temozolomide), the median survival remains approximately 12 – 15 months, with fewer than 25% of patients surviving up to 2 years and less than 10% of patients surviving up to 5 years. We sought to identify a preliminary set of baseline tissue-based DNA and protein features which may reflect growth mechanisms responsible for disease progression, and overall survival (OS).

Methods: Formalin fixed paraffin embedded specimens from 54 patients (44 Astrocytoma (including 14 GBM), 6 oligodendroglioma and 4 mixed) with outcome data and a median follow-up of 3 years were evaluated using dual-color DNA FISH for PTEN and EGFR. A multispectral immunofluorescent (IF) assay was used as previously described (Cordon-Cardo et al., 2007) to evaluate Ki67, PTEN, and P-AKT. Feature performance was univariately assessed using Kaplan-Meier survival curves.

Results: A >10% Loss of heterozygosity (LOH) for PTEN (p-value <0.05) but not EGFR amplification / polysomy was associated with decreased OS in the total cohort. By comparison, the combination of PTEN LOH and EGFR amplification/polysomy in the GBM-only group (n=12, complete data files) was associated with improved survival (p-value 0.03). By quantitative IF, increasing levels of KI67 were associated with shortened OS (p-value 0.004) in the total cohort.

Conclusions: The incorporation of DNA FISH and quantitative IF is useful for developing a biologic baseline for understanding disease course in malignant gliomas. PTEN LOH and EGFR amplification / polysomy in GBM may suggest a pathway mechanism for favorable response.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):A40
Abstract A9: Preclinical modeling of responses and resistance of residual metastatic disease to adjuvant chemotherapy
Authors: Day Chi-Ping , Carter John , Bonomi Carrie , Hollingshead Melinda , Merlino Glenn
Published: January 2011

The questionable predictive power of available preclinical models is explained in part by their failure to adequately replicate the clinical setting. Prevailing human xenograft models do not consider advanced disease, require the use of immunocompromised mice that reconstitute aberrant tumor-host interactions, and employ delayed tumor growth as an endpoint, which has limited clinical relevance. To overcome these deficiencies, we have developed a rapid preclinical model of spontaneous metastasis that more accurately recapitulates clinical procedures for advanced non-small cell lung cancer patients. Lewis Lung Carcinoma tumor tissue that was never adapted to cell culture was stably labeled using lentivirus-encoding bioimaging markers, inoculated into syngeneic C57BL/6 mice, and resected upon reaching a predetermined size. Mice were then treated in a setting akin to post-surgical, first-line adjuvant chemotherapy using cisplatin, paclitaxel and/or antiangiogenic agents. As in the clinic, these drugs were most effective against progression in combination. However, the response of metastases to agents could not be predicted from, and often opposed, their effects on subcutaneous tumors. Time to macrometastatic onset, instead of growth, correlated with mouse survival. Moreover, the most effective adjuvant chemotherapy did not inhibit growth, but rather extended the equivalent of clinical dormancy. These results indicate that metastatic disease is driven by micrometastatic to macrometastatic transition, and resistance to therapies is caused by the failure to maintain clinical dormancy. Identification of the mechanisms involved in these processes will provide new targets of anti-cancer therapies and biomarkers for monitoring therapeutic efficacy. We demonstrate the inadequacies of current preclinical models, and introduce a paradigm for a more clinically relevant model that may more accurately predict therapeutic response in late-stage cancer patients.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):A9
Abstract B12: A novel gene signature associated with response to adoptively transferred T cells in a mouse model of breast cancer
Authors: Martin Michele L. , Chun Hye-Jung Elizabeth , Hsu Janine , Ou David , Jones Steven M. , Watson Peter , Nelson Brad H.
Published: January 2011

Introduction: Adoptive T cell therapy involves the infusion of large numbers of activated, tumor-reactive T cells. In clinical trials, this method often results in mixed responses, wherein some tumor nodules respond while others do not, indicating that the local tumor environment is a critical determinant of response.

Aim: We sought to identify a gene expression signature associated with sensitivity of mammary tumors to adoptive T cell therapy in a mouse model of breast cancer.

Materials and Methods: A transgenic mouse model was used to derive a panel of mammary tumor lines expressing an ovalbumin-tagged version of the HER-2/neu oncogene. Within the panel were tumor lines that reproducibly underwent complete regression (CR), partial regression (PR), or progressive disease (PD) after adoptive transfer of naive, ovalbumin-specific CD4+ (OT-II) and CD8+ (OT-I) T cells. Six representative tumors representing three outcome groups (CR, PR, and PD) underwent Affymetrix gene expression profiling, and results were validated by quantitative PCR.

Results: Gene expression profiling identified four biological pathways associated with outcome following adoptive immunotherapy: leukocyte extravasation, extracellular matrix receptor, GM-CSF receptor, and LXR/RXR receptor pathways. From these pathways, 32 genes that ranked highest for discriminating CR and PD were chosen for further study. The tight junction protein Claudin 4 was expressed at high levels in PD tumors, intermediate levels in PR tumors, and low levels in CR tumors. By contrast, expression levels of the secreted extracellular matrix protein Reelin and the pro-inflammatory cytokine IL-18 were higher in CR tumors compared to PR and PD tumors.

Conclusions: Pathways related to cell junctions, extracellular matrix and inflammation influence the response of mammary tumors to adoptively transferred T cells. Currently, we are modulating expression of these and other genes in mammary tumors to identify regulatory nodes that determine outcome and are amenable to therapeutic intervention. Funding: Canadian Institutes for Health Research, Michael Smith Foundation, BC Cancer Foundation

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B12
Abstract B15: Immunochemotherapy with 5-fluorouracil and interferon-enhances innate and adoptive immune responses against pancreatic cancer in vivo
Authors: Khallouf Hadeel , Serba Susanne , Jäger Dirk , Schmidt Jan
Published: January 2011

Pancreatic cancer has the poorest prognosis of all cancers urging the necessity of new therapeutic approaches. Recent clinical data show encouraging results for combining Interferon-α (IFN-α) immunotherapy with 5-Fluorouacil (5-FU) chemotherapy. However, the role of IFN-α in this immunochemotherapy scheme is still elusive. Here we investigate in vivo the relevance of different immune subpopulations in the anti-cancer immune response mediated by IFN-α in combination with 5-FU.

Luciferase-transfected Panc02 cells were implanted in the pancreas of C57BL/6 mice. Five days later, mice were treated with 5-FU alone, 5-FU + IFN-α or with a vehicle control. In parallel, CD4+ T cells, CD8+ T cells, and NK cells were depleted by neutralizing antibodies. Depletion of CD11c+ dendritic cells (DCs) was performed using transgenic CD11c.DTR mice. On day 21, tumor volume was measured and NK cells were isolated and enriched from spleens of mice then used as effectors against Panc02 cells in a standard chromium release assay. Tumors were frozen for further immunohistochemistry analysis to evaluate the immune cells infiltration. In parallel, we performed flow cytometry analysis to evaluate the effects of different treatments on the expression of MHC-I and NKG2D ligands (MULT-1 and Rae-1) on Panc02 cells.

Our data show that treatment with IFN-α + 5-FU has significantly decreased tumor volume in comparison with control or 5-FU treatments. This decrease in tumor volume was remarkably abolished by depleting CD8+ T cells or NK cells. Furthermore, NK cells isolated from spleens of IFN-α + 5-FU treated mice showed enhanced cytotoxicity towards Panc02 cells. Moreover, IFN-α + 5-FU treatment enhances the expression of MHC-I and NKG2D-ligand MULT-1 on Panc02 cells which could be the possible key for the enhanced recognition and per se killing by CD8+ T cells and NK cells respectively. To conclude, this study gives more insight about the mechanism of action of INF-α in combination with 5-FU and introduces CD8+ T cells and NK cells as main players in the observed anti-cancer immune response.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B15
Abstract B16: Expression of genes involved in PI3K/Akt signaling pathway in arsenic trioxide resistant clones derived from Jurkat cell line
Authors: Arkusz Joanna , Smok-Pieniazek Anna , Komorowska Magdalena , Stepnik Maciej
Published: January 2011

Arsenic trioxide (ATO) is a well-known carcinogen but also an effective cancer therapeutic drug for acute promyelocytic leukemia. Despite intensive research efforts, the molecular mechanisms underlying antileukemic effect of ATO as well as resistance to arsenic therapy appearing during treatment are not well understood. There are data showing that dysregulation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, a central integrating point of survival signals, may be the reason of apoptosis resistance in leukemic cells.

The aim of the present study was to assess expression of 84 genes involved in PI3K/Akt signaling pathway in Jurkat cells exposed to ATO at 1, 2.5 and 5 μM for 16 hrs as well as in ATO resistant clones derived from the cells. First screening of genes was performed using the Human PI3K-AKT Signaling Pathway RT2 ProfilerTM PCR Array (Superarray) and then selected genes were verified in a standard real-time PCR.

Results of our study showed that ATO induced more intensive changes in expression of PI3K/Akt pathway genes in ATO resistant clones than in Jurkat cells exposed to arsenic. Among genes differently regulated in both cell types were these playing a key role in regulating cell growth e.g. GRB10 (growth factor receptor-bound protein 10), FASLG (Fas ligand), CCND1 (cyclin D1). Interestingly, the level of FASLG mRNA was strongly down-regulated in ATO treated Jurkat cells and up-regulated in ATO resistant clones. Expression of CCND1 and GRB10 varied between resistant clones in a concentration-dependent manner. Moreover, concentration-dependent up-regulation of JUN (jun oncogene) was the most characteristic effect induced by ATO in Jurkat cells. Comparing to untreated Jurkat cells, we identified several genes differently regulated in ATO resistant clones (but not in Jurkat ATO exposed cells) that were mainly involved in anti-apoptotic pathways (e.g. IRS1 (Insulin receptor substrate 1), MAP2K1 (Mitogen-activated protein kinase kinase 1), MAPK3 (Mitogen-activated protein kinase 3), RPS6KA1 (Ribosomal protein S6 kinase, 90kDa, polypeptide 1), PRKCB (Protein kinase C, beta 1), PRKCZ (Protein kinase C, zeta) or Akt activation and growth regulation (e.g. PDK1 (Pyruvate dehydrogenase kinase, isozyme 1), MTOR (Mechanistic target of rapamycin), MTCP1 (Mature T-cell proliferation 1)).

In conclusion, our in vitro study identified several targets of ATO that may mediate cytotoxic effects in Jurkat cells. Moreover, the data suggest that the PI3K/Akt signaling pathway plays an important role in the ATO resistance in Jurkat leukemic cells. The work was supported by the Polish State Committee for Scientific Research (grant No PB 2806/B/PO1/2007/33).

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B16
Abstract B22: Beta-arrestin2-CARMA3 signaling axis plays critical roles in lysophosphatidic acid-induced ovarian cancer migration and invasion
Authors: Sun Jiyuan , Cai Jingli , Yumei Feng
Published: January 2011

Ovarian cancer is ranked as the 4th most common type of cancer among women in the United States. Lysophosphatidic acid (LPA) is the diagnostic marker of ovarian cancer. It is significantly elevated in 90% of ovarian cancer patients. Moreover, LPA receptors are aberrantly up-regulated in ovarian cancer patients. LPA is a type of G protein-coupled receptor ligand. In our previous studies, we have demonstrated that a signaling axis consisting of beta-arrestin 2 and CARMA3 (CARD and MAGUK domain-containing protein 3) is required in LPA-induced nuclear factor kappa B (NF-κB) activation in mouse embryonic fibroblast cells. NF-κB is an important transcription factor, which has been proven to mediate LPA-induced ovarian cancer migration and invasion. In this study, we further investigated whether the beta-arrestin2-CARMA3 signaling axis plays a significant role in LPA-induced ovarian cancer cell migration and invasion. Using beta-arrestin 2 and CARMA3 shRNA, we knockdowned their protein expression levels in ovarian cancer cell lines. Consistent with previous reports, we found that down-regulation of beta-arrestin 2 and CARMA3 abolished LPA-induced IKK activity and NF-κB activation in ovarian cancer cells. In addition, in vitro transwell migration and matrigel invasion assays demonstrated that beta-arrestin 2 and CARMA3 shRNA significantly impaired LPA-induced ovarian cancer cell motility and invasiveness. Together, our results provide the evidence that beta-arrestin 2 and CARMA3 serve as critical regulators in LPA-induced, NF-κB-mediated ovarian cancer migration and invasion. Therefore, we speculate that beta-arrestin 2 and CARMA3 may represent attractive therapeutic targets for ovarian cancer and many other types of malignancies.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B22
Abstract B24: Establishment of human primary non-small cell lung cancer xenograft models to test anticancer drugs
Authors: Liu Changnian , Li Wenwei , Li Bin , Liu Yong , Huang Wenpeng , Lou Boliang , Yang Hua
Published: January 2011

Background: Human xenograft tumor models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But such tumor models have a relatively low transplantability and limited correlation with clinical findings. Recently, we have developed human non-small lung cancer (NSCLC) xenograft models by transplanting human fresh lung cancer tissues into nude mice, which have been employed for test of clinically used chemotherapeutical drugs and are under way for new drug chemosensitivity in vivo screening.

Methods: The fresh lung cancer samples were collected from local hospitals. The tumor fragments of 1–2 mm3 were subcutaneously implanted in the flanks of the Balb/c nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 1). In the first passage, tumors derived from male patients were implanted into male mice, and tumors from women were inoculated into female mice. All therapeutic efficacy experiments used female mice. The drugs tested were clinically used drugs for NSCLC including cisplatin (4 mg/kg, q3d x 4), paclitaxel (15 mg/kg, q3d x 4), vinorelbine (2.5 mg/kg, q4d x 4), gemcitabine (120 mg/kg, q4d x 4), and the epidermal growth factor receptor (EGFR) inhibitor erlotinib (50 mg/kg/daily x 14, po).

Results: A total of 42 NSCLC samples were implanted, and 13 tumor models were established (tumor taking rate 31%) for the first passage. The tumor taking rates were higher in the second and third passages (80–100%). Cisplatin, paclitaxel, vinorelbine, and gemcitatbine produced tumor growth inhibition rates of 48–55% regardless of the EGFR mutation status. While erlotinib demonstrated a significant antitumor activity only in the tumors bearing EGFR, which were consistent with their clinical findings. The tumor xenografts' architecture, the cell and histopathological morphology from the three generations mirrored the original patient cancers.

Conclusions: These results suggest that human primary tumor xenograft models provide a unique renewable source of tumor material for test of novel anticancer agents. They may predict more relevant clinical response rates and higher correlation with clinical findings than use of xenograft models established from in vitro long-term cultured cancer cell lines, especially for test of target-oriented therapeutics in new drugs development programs.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B24
Abstract B27: The differential effect of all-trans retinoic acid and fenretinide in regulating differentiation and apoptosis, respectively of liver cancer cell
Authors: Wan Yu-Jui Yvonne , Bushue Nathan , Yang Hui
Published: January 2011

All-trans retinoic acid (RA) and its synthetic analog fenretinide [N-(4-hydroxyphenyl) retinamide] have very different effect on cancer cells. All-trans RA is a powerful differentiation agent and fenretinide is a reactive oxygen species (ROS) inducer that causes apoptosis of various types of cancer cell. It seems that both differentiation and apoptosis effect of these retinoids is mediated through retinoic acid receptor β (RARβ). In addition, both chemicals can induce the level of RARβ mRNA. However, our data showed that all-trans RA was a more effective RARβ inducer than fenretinide. The induction occurred within 30 min upon all-trans RA treatment vs. 3 hrs after fenetinide treatment in Huh7 cells. Moreover, both chemicals induced the expression level of LC3. However, the conversion of LC3-I to LC3-II, a hallmark of autophagy, was substantially higher in all-trans RA than fenretinide treated Huh7 cells. These data suggested the all-trans RA, but not fenretinide, was an effective autophagy inducer. In addition, the level of signaling adaptor p62, which is crucial for activation of death receptors and caspase 8-mediated apoptosis, was significantly lower in all-trans RA than fenrentinide treated Huh7 cells. Consequentially, all-trans RA-mediated autophagy might reduce ROS generation and inhibit apoptosis, whereas fenretinide caused ROS generation and cell death. The role of RARβ in differential regulation of LC3 expression and conversion as well as p62 expression remains to be determined. Taken together, the differential autophagy induction and p62 degradation might account for the different effect of all-trans RA and fenretinide in controlling the fate of liver cancer cells. (This research is supported by NIH grant CA53596.)

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B27
Abstract B28: Epigenetic contribution of Wnt antagonists SFRP1 and DKK1 as prognostic markers in colorectal cancer
Authors: Bapat Bharati , Rawson James B. , Mrkonjic Miralem , Green Roger C. , Gallinger Steve , Younghusband Banfield , McLaughlin John , Knight Julia A.
Published: January 2011

Background: Aberrant Wnt pathway activation is a vital carcinogenic event in colorectal cancer (CRC). DKK1 and SFRP1 encode extracellular inhibitors of canonical and canonical/non-canonical Wnt signaling, respectively, that are frequently silenced by promoter hypermethylation in CRC. Despite their known tumor-suppressive roles, few studies have systematically examined the prognostic/predictive significance of methylation in these genes in tumor development. Using a population-based genetic epidemiological approach, we investigated the methylation status of DKK1 and SFRP1 in a large cohort of primary CRCs and correlations to patient clinicopathological data.

Methods: As part of a Canadian interdisciplinary initiative to study the genetic and environmental determinants of CRC, we accrued a large number of primary colorectal carcinoma cases diagnosed in the province of Ontario, representative of a heterogeneous population (n = 558), and in the province of Newfoundland, representative of a founder population (n = 650). We examined the methylation status of DKK1 and SFRP1 gene promoters in colorectal tumors and matched normal colon tissues using MethyLight assay, a semi-quantitative methylation detection technique. We examined correlations between methylation levels and frequency, and a comprehensive array of patient clinicopathological features such as: age, sex, tumor stage, grade, tumor MSI subtype, and clinical outcome. Statistical analysis was performed using 2-tailed Fisher's exact test, SPSS v16.

Results: Respective DKK1 and SFRP1 methylation frequencies were similar in Ontario (13%, 95%) and Newfoundland (14%, 94%). Methylation was highly tumor specific. DKK1 methylation was a strong predictor of the microsatellite instability (MSI) tumor subtype in Ontario (OR=13.7 [7.8, 24.2], p < 0.001) and in Newfoundland (OR=3.9 [2.8, 5.6] p < 0.001). SFRP1 methylation was a predictor against MSI tumors in Ontario (OR=0.25 [0.15, 0.52], p < 0.001).

Conclusion: Our study highlights the prognostic role of DKK1 and SFRP1 methylation related to CRC tumor subtype and suggests a novel distinction between the relative involvements of different Wnt pathways in microsatellite stable vs. unstable CRC.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B28
Abstract B29: Expression of BARD1 predicts outcome in colon cancer
Authors: Sporn Judith C. , Jung Barbara H.
Published: January 2011

Background: The BARD1/BRCA1 heterodimer has been implicated in several different pathways crucial for maintaining genomic integrity, DNA repair and cell survival. Complex functions have been ascribed to BARD1: It acts as a chaperone that recruits BRCA1 to the nucleus to form an ubiquitin E3 ligase complex involved in DNA repair and chromosomal stability. On the other hand, BARD1 may also promote cell survival by inhibiting BRCA1-dependent apoptosis through nuclear retention of BRCA1. Mutations in the BARD1 gene, as well as aberrant expression of BARD1 splice isoforms, have been associated with poor prognosis in breast and ovarian cancer. Here, we assess the occurrence and role of BARD1 splice isoforms in colon cancer formation.

Methods: Total RNA from primary human colon cancer samples and matched normal colon tissue was extracted and reverse transcribed. Gene specific PCR primers were designed to analyze the occurrence and pattern of BARD1 splice variants in colon cancer. Splice variants were subcloned and sequenced. Protein expression of BRCA1 and BARD1 in paraffin-embedded cancer tissues was detected by immunohistochemistry and correlated with clinicopathologic parameters.

Results: mRNA analysis of primary colon cancers confirmed a specific pattern of BARD1 splice isoforms distinct from normal controls. Certain isoforms appear to be tumor specific. Full length BARD1 is upregulated in non-metastatic cancer samples compared to matched normal tissue, which is in line with its anti-apoptotic function. Interestingly, full length BARD1 is dramatically downregulated in metastatic cancer tissue supporting its role as a tumor suppressor. Immunohistochemical analysis of colon cancer samples confirmed the loss of full length BARD1 in metastatic tissues on the protein level and revealed a significant correlation (p<0.05) between expression levels of BARD1 and outcome, in which low expression of BARD1 is associated with a worse prognosis.

Conclusion: Our results suggest critical roles for the BRCA1 chaperone BARD1 in colon cancer formation. Alternative splice variants of BARD1 commonly occur in colon cancer tissue and expression of full-length BARD1 is differentially regulated along the cancer-metastasis axis affecting cancer outcome. We hypothesize that in colon cancer tissue, BRCA1 function critically depends on BARD1 expression.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B29
Abstract B3: A nested case control study of the polymorphic locus -202 in the promoter region of the IGFBP-3 gene and breast cancer risk
Authors: Macis Debora , Maisonneuve Patrick , Johansson Hariett , Aristarco Valentina , Guerrieri-Gonzaga Aliana , Rotmensz Nicole , Santillo Barbara , Gucciardo Giacomo , D'Aiuto Giuseppe , del Turco Marco Rosselli , Costa Alberto , Veronesi Umberto , Decensi Andrea , Bonanni Bernardo
Published: January 2011

Background: The Insulin-like Growth Factors (IGFs) system has been shown to have an important role in breast carcinogenesis due to its involvement in the regulation of cell proliferation, differentiation and apoptosis. Several studies have found an association between serum concentration of IGF-I and IGFBP-3 - its major binding protein - and breast cancer risk.

A single nucleotide polymorphism in the promoter region of the IGFBP-3 gene (rs2854744), located at position -202 from the transcription start site, results in reduced promoter activity and decreased IGFBP-3 levels. Some evidence of an association between this polymorphism and breast cancer risk has been reported.

Methods and results: We conducted a nested case-control study within the Italian Tamoxifen Chemoprevention Trial, a phase III double-blind placebo-controlled multicentric prevention trial involving 5,408 hysterectomized women aged 35–70 years, who had received either tamoxifen 20 mg/day or placebo for 5 years. After an initial median follow-up of 81.2 months, 79 subjects were diagnosed with breast cancer. Forty-six patients (cases) consented a blood withdrawal as well as 136 controls without breast cancer, randomly selected and matched for age (±2 years), randomization date (±6 months) and participating center.

We genotyped case and control subjects for IGFBP-3 -202A>C with a Taqman real time PCR method for allelic discrimination (Applied Biosystems).

The IGFBP-3 -202A>C genotype frequencies for AA, AC, CC were 46%, 43% and 11% respectively in breast cancer cases and 31%, 48%, 21% in controls. We found a significant trend for a higher frequency of A allele within breast cancer cases compared to unaffected control subjects (Ptrend=0.04). We observed an odds ratio of 0.35 (95% CI 0.12–1.02) for breast cancer in subjects with CC compared to AA genotype, while no further association was found after stratifying for age and treatment group.

Conclusions: We showed that in the analyzed population within the Italian Tamoxifen Chemoprevention Trial, the genotype frequencies for the IGFBP-3 -202 A allele were higher in breast cancer cases compared to unaffected control subjects at variance with other observations. These results suggest that variation at the polymorphic locus -202 in the IGFBP-3 gene might have an influence on breast cancer risk, although further studies are mandatory to clarify the direction of this association and the possible biological meaning and clinical implications.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B3
Abstract B30: Regulation of angiogenesis by the VEGF/miR-101/EZH2 axis
Authors: Smits Michiel , Mir Shahryar E. , Nilsson Jonas , van der Stoop Petra , Niers Anneke , Marquez Victor , Cloos Jacqueline , Breakefield Xandra , Krichevsky Anna , Noske David , Tannous Bakhos , Wurdinger Thomas
Published: January 2011

Rationale: Angiogenesis is a balanced process controlled by pro- and anti-angiogenic molecules of which the regulation is not yet fully understood. Besides classical gene regulation, miRNAs have emerged as post-transcriptional regulators of angiogenesis. In addition, epigenetic changes caused by histone modifying enzymes have emerged as modulators of angiogenesis as well. So far, the interplay between miRNA modulation and histone-modulating enzymes during angiogenesis is unclear.

Objective: To investigate the functions of miR-101 and of its target, the histone methyltransferase EZH2 in endothelial cells and angiogenesis.

Methods and Results: We show that the pro-angiogenic effects caused by VEGF-mediated down-regulation of miR-101 are partly mediated through reduced translational repression of the histone methyltransferase EZH2. In vitro, the sprouting and migratory properties of primary endothelial cell cultures were reduced by inhibiting EZH2 through up-regulation of miR-101, siRNA-mediated knockdown of EZH2, or treatment with 3-Deazaneplanocin A (DZNep), a small molecule inhibitor of EZH2 methyltransferase activity. In addition, we studied the effects of EZH2 inhibition on tumor angiogenesis in vivo. We found that systemic DZNep administration reduced the number of blood vessels in a subcutaneous glioblastoma mouse model, without showing adverse toxicities.

Conclusion: By determining a pro-angiogenic VEGF/miR-101/EZH2 axis in endothelial cells we provide evidence that there is a functional link between growth factor-mediated signaling, post-transcriptional silencing, and histone methylation in the angiogenesis process. Inhibition of EZH2 may prove therapeutical in diseases in which aberrant vascularization plays a role.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B30
Abstract B31: Pharmacogenetics and pharmacoepigenetics of childhood acute lymphoblastic leukemia
Authors: Lopez-Lopez Elixabet , Martin-Guerrero Idoia , Ballesteros Javier , Uriz Javier , de Andoin Nagore Garcia , Piñan M. Angeles , Garcia-Miguel Pura , Navajas Aurora , Garcia-Orad Africa
Published: January 2011

Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer, and still an important cause of cancer-related death in children. Although the introduction of treatment protocols has improved survival, inter-individual differences in drug responses are an important cause of resistance to treatment and adverse drug reactions. These differences can be due to different factors, including genetic polymorphisms and epigenetic variations.

The aim of the present study was to determine if there was a correlation between genetic and epigenetic variation and toxicity and/or outcome during therapy in paediatric ALL patients treated according to the LAL/SHOP protocol (high-dose methotrexate [MTX] and 6-mercaptopurine [6-MP]).

We analyzed genetic polymorphisms and epigenetic variations of 12 genes in paediatric ALL patients: 6 genes of the MTX pathway (MTHFR, TYMS, SHMT1, RFC1, ABCB1 and ABCG2), 1 gene of the 6-MP pathway (TPMT) and 5 genes involved in xenobiotic detoxification (CYP1A1, NQO1 and the GSTs GSTM1, GSTT1 and GSTP1).

Several associations were found between polymorphisms and clinical parameters, such as that of the MTHFR C677T and A1298C polymorphisms and hepatotoxicity and gastrointestinal toxicity respectively.

Our results indicate that polymorphisms in the MTX pathway may be useful as predictors of toxicity and outcome in ALL patients. This project was supported by RTICS (RD/06/0020/0048) and Basque Government (IT-463-07 and 2006111015).

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B31
Abstract B32: DNA demethylation and inhibition of HDAC1 and HDAC2 and is sufficient to reactivate epigenetically silenced tumor suppressor genes
Authors: Geutjes Ernst , Bernards Rene
Published: January 2011

Loss of tumor suppressor gene (TSG) function contributes heavily to both the initiation and progression of cancer and can be the result of mutations in the DNA sequence, but can also be caused by epigenetic disruptions. In contrast to somatic mutations of tumor suppressor genes, epigenetic alterations are essentially reversible. Promoter hypermethylation and histone hypoacetylation play a key role in epigenetic silencing of TSGs. Indeed, the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) and the broad spectrum histone deactelyase (HDAC) inhibitor vorinostat are promising anti-cancer drugs. HDAC inhibitors and DAC are currently explored as combination therapy as they synergistically reactivate epigenetically silenced TSGs and may thus have enhanced therapeutic responses when combined. Although pan-HDAC inhibitors have clear anti-tumor activity in the clinic they induce side effects. In addition, as low expression of some HDACs has been associated with cancer development, their further inhibition may actually contribute to tumor progression. Thus, there is great interest to develop selective HDAC inhibitors. However, it is currently unclear which HDAC(s) to hit.

We thus screened a HDAC siRNA library for their ability to synergize with DAC in reactivation of silenced TSGs. We found that only HDAC1 knockdown synergized with DAC treatment in the re-expression of epigenetically silenced TSGs. Additional knockdown of HDAC2 dramatically enhanced this effect, suggesting that both HDAC1 and HDAC2 are involved in the silencing of TSGs. We found that the HDAC inhibitor MS-275, which only targets HDAC1-3, was also able to synergize with DAC in reactivation of silenced TSGs which confirms our finding that HDAC1&2 are essential for epigenetic silencing of TSGs.

Treatment of cancer cells with DAC and MS-275 resulted in caspase activation which correlated with reactivation of silenced TSGs. We found a strong synergy with DAC and MS-275 in induction of caspase activity and mitochrondrial membrane permeabilization. Collectively, these data show that DNA demethylation and inhibition of HDAC1&2 is sufficient to reactivate silenced TSGs. In addition, these studies suggest that compounds that inhibit HDAC1&2 might be as effective as pan-HDACi as anti-cancer drugs.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B32
Abstract B33: A novel isoform of the major 8p22 tumor suppressor Deleted in Liver Cancer 1 (DLC1) gene with growth suppression function is frequently silenced epigenetically in multiple tumors
Authors: Low John S.W. , Tao Qian , Goh Hwee Koon , Ng Ka Man , Woo Wan Lu , Ambinder Richard F. , Srivastava Gopesh , Shamay Meir , Chan Anthony T.C. , Popescu Nicholas C. , Hsieh Wen-son
Published: January 2011

Aberrant DNA methylation of Tumor Suppressor Gene (TSG) promoter regions, in addition to being a hallmark of cellular transformation, could provide potential tumor markers for early detection and monitoring of treatment effect. In a previous study to identify candidate TSGs relevant in Nasopharyngeal Carcinoma (NPC), our group identified the Deleted in Liver Cancer 1 (DLC1) gene to be epigenetically silenced not just in NPC, but in esophageal, breast and cervical carcinomas also. In this study, we report the cloning and characterization of a novel isoform of the human DLC1 gene with growth suppression function transcribing from a functional alternative promoter and silenced epigenetically in multiple tumors. Designated as DLC1 isoform 4 (DLC1-i4), this novel isoform encodes a protein of 1125 amino acids with a distinct N-terminus as compared to the other known isoforms. DLC1-i4 was expressed ubiquitously in normal tissues and immortalized normal epithelial cells, but significantly down-regulated in multiple tumor cell lines. Promoter DNA methylation was further found in 6/16 (38%) esophageal, 3/4 (75%) cervical, 2/2 (100%) colorectal, 4/4 (100%) NPC cell lines, and in 37/39 (95%) and 12/31 (39%) of primary NPC and esophageal tumors respectively. In contrast, no DNA methylation was found in normal cell lines and tissues. Pharmacological treatment with 5-aza-2′-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B could demethylate the DLC1-i4 promoter and activate transcription, indicating a direct epigenetic mechanism. Ectopic expression of DLC1-i4 had a strong inhibitory effect on the growth and colony formation ability of silenced tumor cell lines. As a whole, our results suggest that transcription of multiple DLC1 isoforms driven off alternative promoters could modulate the activity of the candidate tumor suppressor DLC1 protein in the pathogenesis of these tumors, and should be further tested as a tumor specific molecular biomarker.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B33
Abstract B34: Identifying sources of medical radiation in a population of children diagnosed with acute lymphoblastic leukemia: Single institution study
Authors: Roher Robin L.
Published: January 2011

Purpose of study: Through record review including parental interview author will identify children diagnosed with acute lymphoblastic leukemia at the Children's Hospital of Pittsburgh in the period 1999–2009 who have had a preconception, prenatal or postnatal exposure to medical radiation.

Description of experimental procedures: The necessity of identifying children at increased risk of childhood cancer through envirnomental exposures and genetics is increasing as the diagnosis rate remains fairly constant and even in the case of very successful therapy for children with acute lymphoblastic leukemia the relapse and refractory rates are significant. This study investigates correlation among a regional group of children in the United States through record review and construction and implementation of parental/family questionnaire.

Summary of data: Preliminary study finds a variety of medical radiation exposures, primarily preconception in this cohort. There is a stronger correlation in children diagnosed with T cell acute lymphoblastic leukemia. This data is indicative of the first five years of the study which is still in progress.

Conclusions: This study supports and documents the role of medical radiation in a cohort of children diagnosed with acute lymphoblastic leukemia in a five year period. Extension of the study and careful correlation with relevant genetic data on these identified children will be critical to the longitudinal study and its usefulness.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B34
Abstract B35: Serum inflammatory biomarkers of acute and persistent fatigue in a preclinical breast cancer treatment model
Authors: Wood Lisa J. , Nail Lillian M. , Elsea Collin R. , Druker Brian J.
Published: January 2011

Aims: Cancer treatment related fatigue (CTRF) is a debilitating symptom of cancer chemotherapy. The acute CTRF that occurs during chemotherapy can become persistent lasting for weeks, month, or years after treatment has ended. Although, treatment-related increases in IL-1β and TNF-α have been implicated in CTRF, the role that these cytokines play in CTRF is unclear. The aim of this study was to determine the relationship between chemotherapy-induced IL-1β and TNF-α production and acute and persistent CTRF in a pre-clinical mouse model.

Methods: Blood levels of IL-1β, TNF-α and several other inflammatory biomarkers were measured in acutely fatigued, non-fatigued, and persistently fatigued mice following administration of cytoxan-adriamycin-5-fluorouracil (CAF), a commonly used adjuvant chemotherapy regimen for the treatment of breast cancer, by assessing changes in voluntary wheel running activity as a proxy for fatigue.

Results: Fatigued mice showed increased blood levels of IL-1β, TNF-α as well as several other inflammatory markers including, IL-5, IL-6, IL-7, IL-9, IP-10, G-CSF, KC, MCP-1 and MIP-1β. Importantly, serum levels of these fatigue associated biomarkers were not significantly increased in non-fatigued CAF treated mice. CAF-treated mice with persistent fatigue had elevated serum levels of IL-6, IL-17, and MIP-1α.

Conclusions: This study advances the science of symptom management by providing evidence that treatment induced increases in IL-1β and TNF-α play a role in acute CTRF. Similar temporal analyses between serum inflammatory biomarkers in the clinical setting may lead to a better understanding of the role of immune dysfunction in the etiology of acute and persistent fatigue.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B35
Abstract B36: Induction of IL-6 by breast cancer chemotherapy mediates loss of fat-free lean body mass in tumor-free mice by decreasing systemic IGF-1
Authors: Wood Lisa J. , Elsea Collin R. , Nail Lillian M. , Winters Kerri A. , Druker Brian J.
Published: January 2011

Aims: Cancer patients treated with cytotoxic chemotherapy often experience changes in body composition that occur independent of changes in food intake or physical activity. These changes may impact physical functioning and quality of life. Interleukin-6 (IL-6), an inflammatory cytokine that is up-regulated in cancer patients treated with cytotoxic chemotherapies, plays an important role in the regulation of body composition. The purpose of this study was to determine the role of IL-6 in the regulation of systemic insulin-like growth factor I (IGF-1) levels and treatment induced changes in body composition using a pre-clinical model.

Methods: Changes in body composition and systemic IGF-1 levels were assessed in IL-6−/− mice and their wild type counterparts before and after 4 cycles of cytoxan-adriamycin-5-fluorouracil (CAF) a common adjuvant breast cancer chemotherapy regimen. Daily food intake and physical activity were monitored throughout treatment.

Results: We found that CAF decreased systemic IGF-1 production in an IL-6-dependent manner. Similar to cancer patients, CAF treatment in WT mice led to a reduction in fat free LBM and bone mass but not fat mass (FM). These changes occurred independently of food intake and level of physical activity. In contrast, IL-6 deficient mice were protected from the treatment induced loss of fat free LBM but not bone mass.

Conclusions: These preclinical findings implicate IL-6 mediated reduction in systemic IGF-1 as a possible mechanism underlying the loss of fat free LBM in cancer patients undergoing cytotoxic chemotherapy. Based upon these pre-clinical findings, examination of these relationships in the clinical setting is warranted.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B36
Abstract B38: Cellular uptake of exogenous Arf6 inhibits the proliferation, invasion, and migration of MDA-MB-231 breast cancer cells
Authors: Afroze Syeda , Uddin M. Nasir , Cao Xiaobo , Gizachew Dawit
Published: January 2011

The two isoforms of the Small GTP binding proteins of the ADP riboyslation factor (Arf1 and Arf6), which are involved in membrane trafficking events are extensively studied. As with most members of the ras superfamily, Arf proteins cycle between a GTP-bound and a GDP-bound form. This cycle is regulated by two groups of proteins: GEFs, which facilitate the exchange of GDP for GTP, and GTPase-activating proteins (GAPs), which promote the hydrolysis of bound GTP. Arf1 and Arf6 have distinct cellular functions despite sharing approximately 70% sequence identity. Arf1 regulates the recruitment of vesicle coat polymers, including COPI, to the Golgi apparatus. In contrast, Arf6 is located at the plasma membrane and implicated in endocytotic pathways and vesicle trafficking [1–3]. Recent studies, using mice injected with invasive melanoma cell lines (LOX) that express a dominantly inhibitory Arf6, have shown that Arf6 is an important regulator of tumor growth and metastasis [4]. Our goal is to study is the internalization of exogenous Arf6 and its effect on MDA-MB-231 breast cancer cells in order to explore Arf6 as a therapeutic agent.

Methods and Results: MDA-MB-231 breast cancer cells were incubated for 24 h with either purified Arf6-GTPγS (GTPγS is the nonhydorlysable form of GTP) or Arf6-GDP at various concentrations (60–500 nM) at 37°C and 4°C and the internalization of the proteins were evaluated using western blot analysis and fluorescence microscopy. Both forms of Arf6 proteins were significantly internalized in MDA-MB-231 breast cancer cells, and the internalized proteins inhibited cell invasion, migration and proliferation when compared to PBS (vehicle)-treated cells (p°C and 4°C, internalization mechanisms other than endocytosis must have been involved. We have also observed the internalization of Arf6 in colon and lung cancer cells at 37°C.

Conclusions and Perspectives: Our findings show that when MDA-MB-231 breast cancer cells are incubated with either Arf6-GTPγS or Arf6-GDP in the cell culture media, the proteins are internalized at both 37°C and 4°C. Though further studies are required in order to understand the internalization mechanisms of Arf6, it is known that direct membrane translocation of some peptides including TAT protein is observed in several cell types treated at 4°C. Arf6 is more basic protein (pls in the range of 8.5–9.5) than Arf1 (pls in the range of 6.0–7.0), which might explain why Arf6 is internalized, but not Arf1. The inhibition of proliferation, migration and invasion of the cells might have been induced by defective Arf6 cycling due to the internalized Arf6, which indicates the potential to exploit Arf6 as therapeutic agent for cancer.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B38