bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4320: Separate analysis of the cancer and stroma cell populations from orthotopically implanted cancer cell lines in fluorescent mice
Authors: Jacobsen Hege K. , Sleire Linda , Wang Jian , Svendsen Agnete , Pedersen Paal-Henning , Gullberg Donald , Enger Per Ø.
Published: July 2012

The tumour microenvironment is increasingly recognised as important in most aspects of tumourigenesis, from tumor formation, growth and invasion, to metastasis and priming of distant metastatic sites. It has been estimated that around 50% of a solid tumor mass may be of stromal origin, including cells of the immune system, endothelial cells and fibroblasts, with the latter being the most abundant. The fibroblasts recruited to a tumor are commonly known as cancer associated fibroblasts (CAFs), and are characterized by their heterogeneity of marker expression and origin, making them a challenge to study.

Therefore, we have developed a new strain of mice for the analysis of the tumour microenvironment in xenograft models. The strain was created by crossbreeding dsRed positive mice with severe combined immunodeficient (SCID) mice, and backcrossing on a SCID background until we obtained a homozygous dsRed-SCID inbred strain. As the tumor cells implanted are non-fluorescent, whereas the stromal contributions to the growing tumors are dsRed positive, the two compartments were successfully separated by Fluorescence-activated cell sorting (FACS) after mechanical and enzymatic dissociation.

The dsRed phenotype is visibly red with the naked eye, and fluoresces under a 488 nm dark reader lamp light. The organs have been confirmed dsRed positive by imaging and further confirmed and quantified by RT-PCR. The SCID phenotype was confirmed by immune phenotyping, using markers for B, T, Tc, Th and NK cells. Furthermore, the mice were confirmed immune deficient when they were orthotopicaly grafted with breast (4T1) and lung (A549) cancer cell lines.

Fibroblast enriched populations were isolated from primary A549 tumours, 4T1 tumours and 4T1 tumour metastasis to the lungs, by subsorting the dsRed positive stromal compartment, excluding cells of the immune system and endothelial cells. Preliminary data clearly show distinct tumor and stroma populations in primary breast and lung cancer, as well as in lung metastasis from breast cancer xenografts. Furthermore, when investigating the breast cancers dissociated at different time points from implantation, it is clear that the percentage of stromal cells in the overall tumour mass decreases as the tumour grows. The fibroblasts were further analyzed for marker expression, cell cycle phase distribution and in vitro growth properties. We conclude that this model provides a valuable tool for investigating the stromal compartment of xenograft tumours.

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 4320. doi:10.1158/1538-7445.AM2011-4320
Abstract LB-307: Hyaluronan deposition correlates with poor survival in pancreatic cancer
Authors: Whatcott Clifford J. , Jiang Ping , Watanabe April , Hostetter Galen , Hu Chengcheng , Shepard Michael , Frost Gregory , Hoff Daniel Von , Han Haiyong
Published: July 2012

Hyaluronan (HA) is an extracellular matrix component that plays an important role in a diverse range of cellular processes including proliferation, tissue hydration, cell motility, inflammation, angiogenesis, and malignancy. Increased HA deposition is a characteristic of many desmoplastic tumors. High HA levels have been correlated with poor prognosis in some cancer types, including gastric, colorectal, breast, ovarian, and bladder cancers. Considering the high desmoplastic stromal content of pancreatic tumors, we hypothesized that high HA levels may contribute to poor prognosis or survival in pancreatic cancer. Utilizing a biotinylated HA binding protein-based staining method in a tissue microarray constructed from resected pancreatic tumor tissues, we assessed HA-specific staining in 43 pancreatic ductal adenocarcinoma samples for which patient survival had been followed for more than eight years. We observed HA staining primarily in the peritumoral, or stromal, compartment of the tumor tissue samples. 100% of the patient tumors stained positive in at least some fraction of the tissue area (stroma) for HA. The staining was scored according to the percentage area of HA-positivity, relative to total tissue area. We observed low (<40%) and high (≥40%) levels of stromal HA in 11 and 32 patient samples, respectively. We tested our hypothesis with the log-rank test and found that the survival rate was significantly lower among patients with high HA (P<0.05). The median survival among those patients with high HA was 299 days, and 738 days for those with low HA (the difference in median survival between the two patient groups is 439 days). These results suggest that HA levels in pancreatic adenocarcinoma patients may be predictive of survival. Because HA functions in maintaining high tumoral interstitial fluid pressure, resulting in vascular compression and limited tumor-drug access, our results also suggest that HA may serve as a potential therapeutic target in pancreatic 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 LB-307. doi:10.1158/1538-7445.AM2011-LB-307
Abstract LB-308: Hypoxia affects gene expression and proteome of prostate cancer cells
Authors: Gomez Christian R. , Knutson Gaylord J. , Schreiber Claire A. , Kosari Farhad , Vasmatzis George , Vuk-Pavlovic Stanimir
Published: July 2012

Tumor-associated hypoxia facilitates malignant progression, metastasis and poor prognosis in prostate cancer (CaP); however, effects of oxygen tension (pO2) in CaP are just beginning to be investigated. To establish mechanisms whereby hypoxia enhances malignant properties and survival of CaP and to identify pO2-regulated tumor-associated macromolecules, we propagated CaP cell lines LnCaP, VCaP and DU145 in hypoxia (pO2=2 kPa) and compared them with normoxically grown cells (pO2=20 kPa); hypoxic cells proliferated faster and secreted more vascular endothelial growth factor (p<0.05). Transcriptome studies revealed different gene expression in cells grown in hypoxia relative to those in normoxia. Interestingly, in hypoxic cells transcripts associated with cancer and urologic disease were overexpressed in comparison to normoxic cells (p<0.001) suggesting an association of low pO2 and aggressive features of CaP. Further, we analyzed the transcriptome in primary CaP cells isolated by laser-capture microdissection (LCM) and whole CaP tissue. We compared the data to those from CaP cells cultured at pO2=20 kPa or pO2=2 kPa. Notably, hypoxia increased transcript levels for pyruvate dehydrogenase kinase isozyme 1, nuclear prelamin A recognition factor, glucose phosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase in all three cell lines (p<0.05) to levels comparable to those found in primary bulk tissue and LCM isolated cells (p<0.005). This finding suggests that gene expression in hypoxically cultured cells is more akin to that in tumor cells in situ than are cells grown normoxically. By 2D-gel electrophoresis, we found that change in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Our results suggest that hypoxia affects transcriptome and proteome in CaP cells. In addition, we screened 20 patient sera and 20 healthy control sera for spontaneous antibodies cross-reactive with VCaP cells and found that the sera reacted with numerous proteins, some previously reported to elicit an immune response in CaP patients (e.g., nucleoporin 62 and transitional endoplasmic reticulum ATPase). By this method we detected numerous novel autoantigens (under validation). Currently we are clarifying the pO2 effects on the relationship of transcriptome, proteome and tumor-associated antigens in CaP cells. Support: DOD PC094680 (CRG), Minnesota Partnership for Biotechnology and Medical Genomics, Mayo Clinic Prostate SPORE 5P50CA091956 (FK); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P).

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 LB-308. doi:10.1158/1538-7445.AM2011-LB-308
Abstract LB-31: A snapshot of the expression signature of androgen receptor splicing variants and their distinctive transcriptional activities
Authors: Hu Rong , Isaacs William B. , Luo Jun
Published: July 2012

Objectives: Multiple androgen receptor (AR) splicing variants implicated in ligand-independent AR signaling have been characterized. These variants are expressed in hormone naïve prostate cancers, and elevated in castration-resistant prostate cancers (CRPC), supporting their potential as biomarkers and therapeutic targets for advanced prostate cancer. The diversity and complexity of AR splicing variants however are not fully understood, raising the need to generate a comprehensive expression signature of AR variants, and to address the relative importance of the individual variants that may confer the castration resistant phenotype.

Methodology: A modified RNA amplification method, termed selective linear amplification of sense RNA (SLASR), was developed to amplify all AR transcripts containing AR exon 3. Amplified AR transcripts from CRPC specimens were profiled using expression microarrays with probes tiled across the human AR gene locus. Primers corresponding to novel expression peaks in the AR locus were designed to amplify and decode the splicing junctions. Coding sequences for a subset of selected AR variants were cloned into expression vectors and assessed for their transcriptional activities. Quantitative RT-PCR was used to determine their in vivo expression patterns in an expanded set of clinical specimens.

Preliminary data: Tiling microarray results revealed a molecular portrait of both canonical and cryptic AR exons across the human AR locus. In addition to expression peaks in AR intron 3, a novel AR exon, termed exon 9, was discovered. Exon 9 was spliced into multiple novel AR variants. Different AR splicing variants were functionally distinctive, with some demonstrating constitutive activity while others were conditionally active. Conditionally active AR-Vs may activate AR signaling depending on the cellular context. AR variant functions however did not appear to depend on the full-length AR. AR variants demonstrated patterns of expression change in clinical prostate tumor specimens consistent with their role in castration-resistant progression. This study provided the first unbiased snapshot of the AR variant signature consisting of multiple AR variants with distinctive functional properties. Our results suggest that some AR variants previously considered to be inactive may confer the castration resistance phenotype depending on the cellular context. If further confirmed, the aggregate function of multiple AR variants may compensate for the low abundance of individual variants in clinical specimens relative to the full-length AR.

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 LB-31. doi:10.1158/1538-7445.AM2011-LB-31
Abstract LB-310: FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE™ platform
Authors: Mayer Julie A. , Pircher Tony J. , Mikolajczyk Stephen D. , Cotter Philip D. , Bischoff Farideh Z.
Published: July 2012

Purpose: Determination of HER2 status by immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH) in patients with breast cancer is considered standard practice for selection of treatment options. Though patients presenting with recurrent and/or metastatic disease are often re-evaluated for HER2, biopsy is not often feasible. Thus, circulating tumor cells (CTCs) are an attractive alternative source of tumor tissue for determining HER2 status to enable a more effective course of treatment.

Experimental Design: Twenty to thirty ml of peripheral blood was collected prospectively from 54 patients diagnosed with late stage metastatic/recurrent breast cancer. CTCs were isolated using the microfluidic CEE™ platform. CTC capture was achieved using a cocktail of capture antibodies, followed by detection with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. HER2 amplification was subsequently assessed by FISH on captured CK+/CD45− and CK−/CD45− cells.

Results: CK+/CD45− cells were detected in 43 of 54 cases (80%). Among the 43 cases in which CK+ cells were detected, high concordance (93%) in HER2 status between primary tumor (by IHC and FISH) and CTCs (by FISH) was observed. An overall sensitivity of 95% and a specificity of 92% were obtained using the OncoCEE-BR™ assay.

Conclusions: Recovery of CTCs from peripheral blood using the CEE™ platform is shown to be efficient and suitable for FISH-based testing. In addition, HER2 FISH on recovered CTCs is proven to be sensitive and accurate.

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 LB-310. doi:10.1158/1538-7445.AM2011-LB-310
Abstract LB-313: Development of a non-invasive, multi-analyte assay for the triage of patients presenting with hematuria
Authors: Fernandez Cecilia A. , Millholland John M. , Zwarthoff Ellen C. , Loughlin Kevin R. , Feldman Adam , Karnes R. Jeffrey , Shuber Anthony P.
Published: July 2012

Introduction

The presence of blood in the urine (hematuria) is one of the hallmark symptoms of bladder cancer. Thus, the current best practice policy suggests that patients with hematuria undergo cystoscopic examination. However, hematuria can be present in healthy individuals as well as in individuals with various non-life threatening conditions such that a majority of patients with blood in the urine (∼95%) who are screened by cystoscopy do not have bladder cancer. In addition, recent studies have found that a majority of patients with hematuria are not referred on to a specialist for further invasive tests, despite the guidelines. This suggests that the prevalence of cancer in this population may be lower than 5%, making screening by cystoscopy inefficient and, in a large number of cases, unnecessary. We are developing a non-invasive diagnostic assay utilizing urinary protein and DNA biomarkers to triage those patients who do not have cancer from those who should go on to be existing invasive screening modalities.

Methods

Urine samples were obtained from 48 cancer patients and 256 patients who were evaluated for hematuria but who did not have cancer upon cystoscopic evaluation (Hem+/Cysto−). Twist1 and Nid2 methylation status was assessed using methylation-specific PCR primers and FGFR3 mutational status was determined by quantitative PCR. Matrix Metalloproteinases (MMPs) levels were determined by ELISA.

Results

Using a novel combination of these DNA and protein markers we identified with high confidence (99% negative predictive value) those patients who do not have cancer and who could be excluded from further testing. Cutoffs could be adjusted such that at 94% sensitivity, 65% of patients who do not have cancer could be excluded.

Conclusions

The Multi-Analyte Diagnostic Readout (MADR) assay described here combines the best performance characteristics of protein and DNA biomarkers into one assay for optimal clinical performance. Using this approach, the detection of FGFR3 mutations and Twist1 and Nid2 methylation in the urine of hematuria patients undergoing screening for bladder cancer effectively increases the sensitivity and negative predictive value at an established MMP protein cutoff. This non-invasive urinary diagnostic assay could lead to the more efficient triage of hematuria patients by identifying those patients who do not have cancer and could be excluded from receiving invasive procedures.

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 LB-313. doi:10.1158/1538-7445.AM2011-LB-313
Abstract LB-316: Association of Bcl2 family gene polymorphisms with response to and toxicity of platinum-based chemotherapy in non-small-cell lung cancer patients in a Chinese population
Authors: Qian Ji , Han Wu Qi , Ru Lu Da , Yi Wei Qing
Published: July 2012

PURPOSE: The Bcl2 family members have dual functions in apoptosis. Platinum agents cause genetic lesions and cell death that may be modulated by the normal pathway of apoptosis regulated by the Bcl2 family genes. We hypothesized that genetic polymorphisms of these genes have an impact on the outcome of platinum-based chemotherapy. The aim of our research was to assess whether single nucleotide polymorphism (SNPs) of BAD, BAX, PUMA, BID, and BCL2 in a pathway of regulating apoptosis affect the response to and toxicity in patients with NSCLC treated with platinum-based chemotherapy.

METHODS AND METERIALS: We genotyped 64 SNPS of 5 genes by Illumina Custom Designed Chip in 1004 patients with advanced NSCLC treated with platinum-based chemotherapy. We investigated the response and the toxicity of patients after platinum-based chemotherapy. The genotyping data were analyzed by the Sofeware of SPSS12.0, PHASE2.1 and Haploview.

RESULTES: A SNP of BID (rs5746474) was significantly associated with the hematological toxicity. Compared with the wild-type GG genotype, the variant AA genotype was associated with significantly increased risk to hematological toxicity (adjusted OR: 2.4344, 95%CI=1.4084–4.2077, p=0.0014) in a recessive model (adjusted OR: 2.5241, 95% CI = 1.4839–4.2933, P=0.0006). In the allelic analysis, a SNP of BCL2 (rs8099575) showed an effect on gastrointestinal toxicity (adjusted OR: 0.3041, 95% CI=0.1524–0.6.68, P=0.0007). Several haplotypes of these genes were associated with the response and toxicity; however, after Bonfernoni correction, no association between haplotypes and clinical outcome remained.

CONCLUSION: Genetic polymorphisms in the BID and BCL2 genes may have an impact on the response to and toxicity of platinum-based chemotherapy in patients with NSCLC, which may be prognostic biomarkers of chemotherapy in NSCLC patients, if validated in larger studies.

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 LB-316. doi:10.1158/1538-7445.AM2011-LB-316
Abstract LB-319: Microarray CGH profiling predicts survival after a diagnosis of epithelial ovarian cancer and identifies novel candidate therapeutic targets
Authors: Jones Christopher , Morris James , Sher Tanya , Ghani Sumera , Jacobs Ian J. , Hogdall Estrid , Blaakær Jan , Kruger-Kjaer Susanne , Christensen Lise , Hogdall Claus , Ramus Susan , Gayther Simon , MacKay Alan
Published: July 2012

There is a need to identify novel therapeutic targets and better prognostic markers for ovarian cancer. Previous studies have shown that somatic genetic alterations are associated with clinicopathological features of cancers, and array comparative genomic hybridisation (aCGH) analysis is an excellent tool for providing detailed genetic profiles of a tumour from archival material. In this study we performed aCGH analysis on 116 primary stage III/IV serous ovarian adenocarcinomas and used a combined univariate and iterative SVM-based approach to identify regions of chromosomal alteration whose copy number status is associated with survival. Cases were stratified according to survival probability (Pr), identifying 39 good survivors (Pr<0.33) and 39 poor survivors (Pr>0.67). We then identified genomic regions of either common gain/loss in all 116 cases, or in which copy number status differed between the extremes of survival. A sub-set of data from these regions in the 78 cases at the extreme ends of survival was supplied to an iterative Support Vector Machine (SVM) routine, which ranked regions by their contribution to accurate survival classification and refined their boundaries. Hierarchical cluster analysis confirmed the potential of the minimal intervals of these regions to predict survival. A SVM was then validated with independent data and shown to be able to accurately classify survival. The highest-ranked chromosome regions contain candidate genes that may represent prognostic markers and/or therapeutic targets for ovarian cancer. One region, containing the CCNE1 gene, is frequently amplified in tumours and has recently been shown to be a prognostic marker for ovarian cancer in other studies. Other regions contain genes involved in pathways that stimulate ovarian cancer cell growth, motility and drug resistance. This study demonstrates the power of tumour profiling in cancer samples to identify prognostic signatures and clues to the underlying biology of tumour development, if the study design is rigorous in the selection of tightly stratified patient subgroups and in the application of the correct computational methodologies. These results require validation in larger sample sizes; but the ability to perform whole genomic profiling in formalin fixed tissues and the establishment of consortia to bring together multiple studies (e.g. the international ovarian cancer association consortium) makes this possible.

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 LB-319. doi:10.1158/1538-7445.AM2011-LB-319
Abstract LB-331: Protein expression signatures of epidermal growth factor receptor inhibition
Authors: Myers Matt , Liebler Daniel C.
Published: July 2012

We have applied standardized shotgun and multiple reaction monitoring (MRM) proteomic platforms to detect protein expression changes associated with epidermal growth factor receptor (EGFR) stimulation and inhibition in A431 carcinoma cells. These experiments compared proliferating, stimulated (30 nmol EGF, 4h) with cells co-treated with EGF and either cetuximab (10 μg/ml) or gefitinib (500 nmol). The analyses are done by shotgun proteomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS), using isoelectric focusing of peptide mixtures, followed by reverse phase LC-MS/MS on a Thermo LTQ instrument. Protein inventories (≥2 peptides per protein at 5% peptide FDR) for each set were approximately 3800 proteins at < 3% protein FDR. Differential protein expression was estimated by comparison of spectral count data with a generalized linear modeling (GLM) approach. EGF stimulation induced expression changes in 148 proteins. Effects of the inhibitors on this “stimulation signature” were then determined by GLM comparisons of spectral count data for cetuximab- and gefitinib-treated cell proteomes. These analyses identified 13 proteins whose EGF-induced expression was reversed by both drugs, 15 proteins reversed by cetuximab and 57 proteins reversed by gefitinib. Targeted MRM analysis verified differential expression of the 13 proteins reversed by both drugs, which comprise an “EGF inhibition signature”. These include the transcription factors c-Jun and JunD and the cell cycle inhibitor Cip1, which were upregulated by EGF and the protein CCDC50, which was decreased by EGF. MRM analyses also identified EGF-induced protein expression changes that were selectively reversed by either cetuximab or gefitinib. Serum deprivation, rather than EGF stimulation may account for a subset of the 106 protein changes that were not reversed by inhibitors in EGF treated cells. Our analyses also identified 87 and 149 proteins that were not affected by EGF, yet were significantly altered by cetuximab and gefitinib, respectively. Activation of the EGFR axis is typically measured via the phosphorylation status of EGFR tyrosine kinase substrates and downstream targets, but these measurements are difficult to achieve in tissues due to the low abundance of phosphorylated protein forms and the challenge of preserving phosphorylation status during sample preparation. Protein expression changes directed by EGFR activation may provide more robust measures of EGFR activation and inhibition. Ongoing studies will test the hypothesis that an EGF inhibition signature provides an early index of therapeutic responses to EGFR-targeted cancer therapies. (Supported by NIH Grant U54CA126479)

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 LB-331. doi:10.1158/1538-7445.AM2011-LB-331
Abstract LB-334: Imaging caspase dependent cell death as a surrogate for efficacy of cancer therapeutics
Authors: Galbán Stefanie , Lafferty Michael , Sharkey Lisa M. , Schinske Katrina A. , Merchant Rahim F. , Ross Brian D. , Rehemtulla A
Published: July 2012

Induction of cell death in malignant lesions still represents the major goal in the treatment of cancer patients. Imaging of apoptosis would enhance the development of optimal dosing, schedule and combination therapies. Here we used a luciferase biosensor (Promega) and developed a bioluminescent assay system with optimized signal to noise for the detection of cell death in vitro and in living animals. Using D54-MG cells stably expressing the reporter, we demonstrate that TRAIL treatment resulted in a 100–200 fold induction of bioluminescence activity which correlated with cell death as demonstrated by increased cleavage of Caspase-3. Furthermore inhibition of apoptosis using the pan caspase inhibitor Z-VAD-FMK resulted in inhibition of reporter activity. The utility of this cell death reporter was further demonstrated in human glioma xenografted tumors wherein induction of apoptosis was correlated with reporter activation. The high signal to noise and dynamic range of reporter activity provides for a sensitive and quantitative surrogate for evaluation of experimental therapeutics. In addition, the ability to image repeatedly provides a unique opportunity to understand the dynamics of cell death in response to specific drugs or combination therapies. We are also investigating the application of this technology in a high throughput screening of compound and targeted siRNA libraries in diverse cells and in pre-clinical cancer mouse models.

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 LB-334. doi:10.1158/1538-7445.AM2011-LB-334
Abstract LB-335: DHODH regulates transcriptional elongation during melanoma evolution: insights from zebrafish screening
Authors: White Richard M. , Zon Leonard I.
Published: July 2012

Melanoma cells are derived from the neural crest, a transient, multipotent tissue present only during embryogenesis. Using zebrafish models, we aimed to determine whether these embryonic neural crest gene programs would make therapeutic targets in melanoma. Transgenic zebrafish expressing BRAFV600E under the mitf promoter develop melanoma when crossed with p53 mutants. From the earliest stages of transformation, and throughout overt tumorigenesis, these tumors express multiple embryonic neural crest progenitor markers such as SOX10, EDNRB and MITF. Human melanomas were similar: 50–75% expressed progenitor markers, but only 15% expressed the differentiation marker DCT. We performed a chemical genetic screen in zebrafish embryos to identify suppressors of the neural crest gene program to determine their therapeutic utility. Of 2000 small molecules, we found one class, inhibitors of the enzyme DHODH, that led to a complete abrogation of neural crest development during embryogenesis. Treated animals were essentially albino, devoid of all derivative melanocytes that eventually give rise to melanoma. Developmentally, DHODH suppression exerts this effect by blocking transcriptional elongation of genes such as MITF, in a manner dependent upon the elongation factor SUPT5H. In melanoma cells, we measured transcriptional elongation after DHODH inhibition via RNA pol II chromatin immunprecipitation coupled with massively parallel sequencing (ChIP-Seq). Similar to embryonic development, inhibiting DHODH leads to a defect in transcriptional elongation at select loci. We then tested whether leflunomide, a DHODH inhibitor approved for rheumatoid arthritis, could block melanoma growth. Either alone, or in combination with the BRAFV600E inhibitor PLX4032, leflunomide led to a dramatic decrease in melanoma growth in vitro and in vivo. These studies suggest that modulating the neural crest identity of melanoma cells is a distinct therapeutic avenue that is likely to cooperate with inhibiting oncogenic mutations such as BRAFV600E. We plan to extend this work into the clinical trial setting, highlighting the rapidity with which discoveries in zebrafish can be translated into the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-335. doi:10.1158/1538-7445.AM2011-LB-335
Abstract LB-338: Stabilisation of MAD2 expression through inhibition of miR-433; A novel mechanism for restoring chemo-responsiveness in ovarian tumours
Authors: Furlong Fiona M. , Phelan Sine , McGoldrick Alo , Maguire Aoife , Fitzpatrick Patricia , O'Toole Sharon , Shiels Orla , O'Leary John J. , McCann Amanda
Published: July 2012

Epithelial ovarian carcinoma (EOC) is characterised by late diagnosis and recurrences, both of which contribute to the high morbidity and mortality of this cancer. Unfortunately, EOC has an innate susceptibility to become chemo-resistant. Specifically, up to 30% of patients may not respond to current standard chemotherapy (paclitaxel and platinum in combination) and of those who have an initial response, some patients relapse within a few months. Therefore, in order to improve patient outcome it is crucial to establish what factors influence a patients' individualised response to chemotherapy. We analysed MAD2 protein expression in a patient cohort of 35 ovarian tumours and a panel of 5 ovarian cancer cell lines. We have demonstrated that low nuclear MAD2 expression intensity was significantly associated with chemo-resistant ovarian tumours (p=0.0136). Moreover, in vitro studies of the 5 ovarian cancer cell lines revealed that reduced MAD2 expression was associated with paclitaxel resistance. In silico analysis identified a putative miR-433 binding domain in the MAD2 3′UTR and expression profiling of miR-433 in the ovarian cancer cell lines showed that low MAD2 protein expression was associated with high miR-433 levels. In vitro over-expression of miR-433 attenuated MAD2 protein expression with a concomitant increase in cellular resistance to paclitaxel. Over-expression of a morpholino oligonucleotide that blocks miR-433 binding to MAD2 3′UTR stabilised MAD2 protein expression and protects from miR-433 induced degradation. Furthermore, miR-433 expression analysis in 35 ovarian tumour samples revealed that high miR-433 expression was associated with advanced stage presentations (p=0.0236). In conclusion, ovarian tumours that display low nuclear MAD2 intensity are chemo-resistant and stabilising MAD2 expression by antagonising miR-433 activity is a potential mechanism for restoring chemo-responsiveness in these tumours.

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 LB-338. doi:10.1158/1538-7445.AM2011-LB-338
Abstract LB-349: Differential expression of microRNAs in oligodendrogliomas of different grades of malignancy
Authors: Serafini Luciano Neder , Panepucci Rodrigo A. , Tirapelli Daniela P. C. , Oliveira Viviane C. , Monteiro Patricia , Simoes Deise L. , Silva Wilson A. , Carlotti Carlos G.
Published: July 2012

MicroRNAs (miRNAs) are large tiny regulatory RNAs found ubiquitously in plants, insects and mammals. More recently, miRNAs have also been implicated in oncogenesis, acting as tumor suppressors or oncogenes. The role of miRNAs in glioma progression has been intensively investigated chiefly in glioblastomas, the most prevalent malignant glioma. However, there are few data regarding to miRNA expression in oligodendrogliomas. The main goal of this study was to evaluate miRNAs and mRNAs that are differentially expressed in a series of grade II and III oligodendrogliomas (7 per group, with duplicates) in order to obtain miRNAs that correlate with their putative target-mRNAs. After microdissection and diagnosis confirmation, miRNAs and mRNAs expression were evaluate using microarray-based expression profiling platforms (723 transcripts and 41,000 genes, respectively) at same tumor specimens. As controls, samples of temporal lobe white matter from patients operated for clinical intractable complex partial seizures were used (n = 16). Fifteen and 29 miRNAs transcripts were relatively hyper- and hypoexpressed in anaplastic oligodendrogliomas; however, after matching of miRNAs and putative target-mRNAs expressions, only 4 miRNAs transcripts remained overexpressed in which target genes have pivotal role in key cell signaling pathways. Similarly, 20 of 29 miRNAs transcripts remained down regulated. Of these, 4 miRNAs that were previously described as to be involved in cell differentiation in embryonic stem cells (ESC) are being validated through their target genes by real time PCR. Herein, we were able to identify putative oncogenic miRNAs that can be operating in oligodendrogliomas progression, expanding our knowledge about tumor biology and disclosing possible molecular targets for treatment proposals.

Support: FAPESP (Grant Proc. No. 05972–2)

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 LB-349. doi:10.1158/1538-7445.AM2011-LB-349
Abstract LB-422: Photodynamic ablation of lymphatic vessels and intralymphatic cancer cells prevents metastasis
Authors: Tammela Tuomas , Saaristo Anne , Holopainen Tanja , Ylä-Herttuala Seppo , Andersson Leif C. , Virolainen Susanna , Immonen Ilkka , Alitalo Kari
Published: July 2012

The dissemination of tumor cells (metastasis) from the primary tumor is the principal cause of death in cancer patients. The lymphatic vessels serve as a key conduit for metastasizing tumor cells, which often colonize the lymph nodes first. Although the primary tumor and lymph nodes invaded by tumor cells are removed during surgery, tumor cells inside lymphatic vessels are not removed. Here, we demonstrate tumor cell growth inside lymphatic vessels in mice bearing mouse melanoma tumors or human lung tumors and in a human melanoma patient, suggesting that these in-transit tumor cells give rise to metastases. We selectively destroyed lymphatic vessels using photodynamic therapy with the benzoporphyrin derivative verteporfin in mice and pigs. Destruction of tumor-draining lymphatic vessels also eradicated intralymphatic tumor cells, and prevented in-transit metastasis of mouse melanoma cells and subsequent relapse. Photodynamic therapy, when combined with anti-lymphangiogenic therapy, prevented further tumor invasion of lymphatic vessels and may provide a way to eradicate in-transit tumor cells in patients.

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 LB-422. doi:10.1158/1538-7445.AM2011-LB-422
Abstract LB-427: Unique properties of hepatocarcinogenesis-resistant DRH rat hepatocytes linked or not linked to the Drh1 locus on rat chromosome 1
Authors: Hashimoto Norikazu , Yamamoto Masahiro , Miyakoshi Masaaki , Tanaka Hiroki , Ogawa Katsuhiro
Published: July 2012

DRH rats were established by the selective mating of Donryu rat progeny that exhibited resistance to hepatocarcinogenesis. DRH rats show smaller and fewer preneoplastic hepatocytic lesions than ordinary rats after treatment with hepatocarcinogenic regimens, and these traits have been assigned to two major genetic loci, Drh1 on chromosome 1 and Drh2 on chromosome 4. The DRH hepatocytes have the unique properties such as low proliferation after lead nitrate (LN) treatment, resistance to hepatotoxic chemicals and small cell size. If some of these properties are linked to the Drh1 or Drh2 loci, the properties may be related to the mechanism(s) of the hepatocarcinogenesis-resistance in DRH rats. In this study, to explore what properties are linked to the Drh1 locus, we compared hepatocytes of DRH.F344-Drh1 rats, a congenic DRH strain in which the DRH chromosomal region including the Drh1 locus was substituted by that of F344 rats, were compared with hepatocytes of Donryu, DRH and F344 rats. After LN treatment, although DRH hepatocytes proliferated at low rate, DRH.F344-Drh1 hepatocytes proliferated at high rate comparable to Donryu and F344 hepatocytes. In addition, the DRH.F344-Drh1 livers showed high p38 MAPK dephosphorylation after LN treatment like Donryu and F344 livers, while such p38 MAPK dephosphrylation was not seen in the DRH liver. However, increase of TNF-α, IL-6 and Cox2 mRNA and NFκB/Stat3 activation in the liver was almost at the same degree in Donryu, DRH and DRH.F344-Drh1 rats, suggesting that the low responses to LN in the DRH hepatocytes were not dependent on low cytokine/Cox2 activation but rather due to the inherent properties of DRH hepatocytes. After 200 mg/Kg diethylnitrosamine (DEN) or 5 ml/Kg CCl4 treatment, severe hepatic injury occurred in DRH.F344-Drh-1 rats like Donryu and F344 rats, while its degree was much less in DRH. On the other hand, the DRH.F344-Drh1 as well as DRH hepatocytes exhibited lower proliferation after the EGF or HGF treatment in vitro than Donryu and F344 hepatocytes. Furthermore, the nuclear size of DRH.F344-Drh1 hepatocytes was comparable to that of DRH hepatocytes, which was smaller than those of Donryu and F344 hepatocytes. These findings indicate that the Drh1 locus is linked to the low responses to LN and hepatotoxin resistance in DRH hepatocytes, but not to low proliferation in vitro and small nuclear size. (Supported by the grants from the Japanese Ministry of Education, Science, Culture and Sports.)

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 LB-427. doi:10.1158/1538-7445.AM2011-LB-427
Abstract LB-433: Examination of the oncogenic potential of XMRV in prostate epithelial cells
Authors: Comeaux Brennen , Zolochevska Olga , Figueiredo Marxa L.
Published: July 2012

Xenotropic murine leukemia virus-related virus (XMRV) is a retrovirus closely related to murine leukemia viruses, which cause a variety of cancers and immunological and neurological diseases in mice. Whether XMRV plays a role in cancer and other diseases remains controversial; some studies show a possible association, but there is currently no evidence that the virus is a causal agent for disease. Our hypothesis was that the 3′ long terminal repeat (LTR) from the XMRV genome is responsible for its oncogenic activity in prostate cells. We chose three regions from the XMRV 3′ LTR and tested the potential of these regions in transactivating a cellular gene promoter whose homolog is transactivated by MMLV in mouse cells. We examined the transactivation potential in pre-cancerous and a panel of cancerous prostate epithelial cells. Our results showed that for certain 3′LTR regions induce nearly full transactivating activity, while others might comprise inhibitory regions. Therefore, to better understand the mechanism by which the XMRV 3′ LTR regions might be inducing or inhibiting transactivation, we tested their ability to transactivate cellular genes related to transformation. We used as reporters plasmids containing binding sites for cellular transcription factors. The results showed several similarities in transactivation, suggesting common activation pathways for 3′LTR transactivating activity. Finally, we tested if XMRV infection could enhance oncogenicity potential of prostate precancerous and cancerous cell lines. We examined this in vitro by assessing the rate of colony formation in soft agar, and in vivo, using xenograft assays. The results suggested that cell lines infected with XMRV had enhanced in vitro colony formation, as well as enhanced in vivo growth, however, addition of another tumorigenic genetic alteration was required in conjunction with XMRV-infection to induce enhanced tumor growth. Our data suggests that the XMRV 3′LTR may have a transactivating activity on cellular genes, and that XMRV might have a transforming activity in prostate cancer cells, most likely stimulating promotion/progression of tumorigenesis, and not ‘initiation’ of tumorigenesis. These results are highly novel since they represent for the first examinations of the role of XMRV in tumorigenesis of prostate epithelial cell lines in vitro and in vivo and examinations of the 3′LTR context of the virus upon transactivation of cellular genes as a potential mechanism of cellular transformation.

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 LB-433. doi:10.1158/1538-7445.AM2011-LB-433
Abstract LB-435: Regulation of emmprin in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells: a novel strategy developed against KSHV-related diseases
Authors: Qin Zhiqiang , Dai Lu , Bratoeva Momka , Toole Bryan , Parsons Chris
Published: July 2012

Purpose: KSHV is a common etiologic agent for cancers arising in the setting of immune suppression, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We recently reported that the KSHV-encoded latency-associated nuclear antigen (LANA) upregulates expression of the multifunctional membrane glycoprotein emmprin (Extracellular Matrix MetalloPRoteinase Inducer) and associated cell invasiveness and colony formation (Qin et al. Cancer Res. 2010; 70(10): 3884–9). The purpose of this study was to determine mechanisms for KSHV/LANA-induced transcriptional activation of emmprin, and whether these interactions affect emmprin-mediated signal transduction.

Experimental Design: We used co-IP, confocal microscopy, gene silencing, and overexpression of LANA deletion mutants to whether specific transcription factors (TF) interact with LANA and mediate LANA-initiated emmprin activation, as well as putative sites of LANA-TF interactions. Pharmacological inhibitors, gene transfer, immunoblots, qRT-PCR, ELISA and transwell assays were performed to assess how LANA-TF interactions impact emmprin-dependent intracellular signal transduction and downstream pathogenesis.

Results: We found that Sp1, a Zn finger TF, mediates LANA activation of emmprin transcription/protein expression and cell invasiveness. The N-terminal domain of LANA, containing a nuclear localization sequence, is necessary and sufficient for Sp1 activation of emmprin and co-localizes with Sp1 in the nucleus. Interestingly, we also found that emmprin-dependent activation of pro-migratory cytokines/chemokines and endothelial cell invasiveness following de novo KSHV infection is dependent upon KSHV induction of PI3K/Akt and ERK signaling, but not NF-κB pathway. Conclusions: LANA interactions with Sp1, and KSHV induction of Akt- and MAPK-dependent signaling, initiate transcriptional activation of emmprin and emmprin-dependent activation of pro-migratory cytokines release and cell invasiveness following KSHV infection. Ongoing experiments to clarify regulatory networks for emmprin activation in KSHV-infected cells may lead to identification of novel therapeutic targets for KSHV-related diseases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-435. doi:10.1158/1538-7445.AM2011-LB-435
Abstract LB-437: ICA-1: A novel PKC-i inhibitor inhibits the proliferation of prostate cancer cells
Authors: Bhayankara Aravind P. , Patel Rekha , Win-Piazza Hla , Pillai Prajit , Acevedo-Duncan Mildred E.
Published: July 2012

Prostate cancers are highly lethal tumors and besides skin cancer, prostate cancer is the most common type of cancer among men in the United States. Approximately one third of men diagnosed with cancer each year will have prostate cancer. Certain prostate cancers are highly lethal tumors due to the emergence of therapy-resistant prostate cancer cells. PKC-iota (PKC-ι) is an oncogene and an atypical protein kinase C isoform that confers resistance to drug-induced apoptosis in cancer cells (J. Biol. Chem. 272; 44: 27521–27524). PKC is of special interest since it is overexpressed in prostate cancers (Proc Natl Acad Sci U S A. 106:16369–16374). The objective of the study was to investigate the effects of inhibiting PKC-ι on the proliferation of prostate cancer cells in-vitro. The methodology involved treating androgen sensitive LnCap cells and androgen independent Du-145 with the novel PKC-ι inhibitor [1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy) methyl]cyclopentyl]-[1R-(1α, 2β, 3 β, 4 α)] (ICA-1) that targets a unique sequence (amino acid residues 469–475) in the catalytic domain of PKC-ι. Cell viability and number was quantified 24–72 hours following addition of either vehicle control or ICA-1 by Trypan blue exclusion. Additional ICA-1 was not added to the cells following the initial treatment. Results showed that at 24 hours post treatment, ICA-1 decreased the proliferation of LnCap cells by 46% (10 μM) and 83% (20 μM). ICA-1 reduced the proliferation of Du-145 cells at 24 hours by 43% (10 μM) and 51% (20 μM). The overexpression of PKC-ι in prostate cancer and the anti-proliferative effects of ICA-1 may translate into using PKC-i detection as a biomarker to identify patients that may benefit from anti-PKC-ι therapy with ICA-1 for personalized medicine.

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 LB-437. doi:10.1158/1538-7445.AM2011-LB-437