bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B39: Molecular diagnostics strategy to identify hereditary non-polyposis colorectal cancer patients
Authors: Kostina Olga , Rebane Egle , Anderson Wally , Kask Martin , Valkna Andres
Published: January 2011

Introduction: Hereditary non-polyposis colorectal cancer (HNPCC), also called Lynch syndrome, is an autosomal dominantly inherited syndrome predisposing to the early development of cancers of colon, rectum, endometrial, small bowel and urinary tract and accounts for ∼5% of all colon cancer cases. Molecular investigations have shown that most HNPCC families are associated with constitutional mutations in a class of genes (called MLH1, MSH2, MSH6, PMS2 and probably others) involved in DNA mismatch repair. MLH1 and MSH2 genes account for approximately 90% of HNPCC families identified germline mutations. Most of these mutations are point mutations and small insertions or deletions. The large genomic deletions in MMR genes and germline epimutations in MLH1 explain a proportion of point mutation-negative families suspected of Lynch syndrome. At the same time the methylation in promoter of MLH1 gene in colon tissue frequently causes MLH1 protein loss in sporadic tumors. The aim of the study is to characterize the mismatch repair gene mutations of Estonian hereditary non-polyposis colorectal cancer families and to improve the screening strategy.

Methods: A systematic analysis of 75 unrelated patients from suspected HNPCC families in Estonia was performed by immunohistochemistry, microsatellite instability, and gene mutation and methylation detection.

Results. The study is still continuing. Systematic and comprehensive analysis of samples from 75 HNPCC-suspected patients is proceeding using various methods.

Conclusion: Based on traditional molecular genetics, combined with epigenetics, multiple detection methods can accurately diagnose HNPCC. As the results of the study, the precise molecular diagnostic scheme for diagnosing HNPCC will be worked out for clinical use in Estonia.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B39
Abstract B4: A microRNA screen to identify modulators of sensitivity to Bcl-2 inhibitor ABT-263
Authors: Semizarov Dimitri , Lam Lloyd , Lu Xin
Published: January 2011

Evasion of apoptosis is a known feature of cancer cells. One mechanism of deregulating the apoptotic pathway is through overexpression of anti-apoptotic Bcl-2 family members. ABT-263 is a first-in-class Bcl-2 family inhibitor that restores the ability of cancer cells to undergo apoptosis. However, many cancer cells are resistant to ABT-263 due to high levels of a Bcl-2 family member Mcl-1, which is not targeted by the drug. Mcl-1 expression is known to be regulated transcriptionally, translationally, and through proteosome-mediated degradation. Recently, Mcl-1 expression was shown to be affected by microRNAs (miRNAs). In order to identify miRNAs that modulate the sensitivity of cancer cells to ABT-263, we screened a library of 810 human miRNA mimics in HCT-116 cells in the presence of ABT-263. The screen revealed 19 miRNAs that sensitize HCT-116 cells to ABT-263. Fifteen of these miRNAs were also shown to sensitize CHL1 melanoma cells to the same agent. We further evaluated twelve of the strongest sensitizers in these cell lines. We found that these sensitizers induced apoptosis only in the presence of ABT-263. In addition, while all twelve of these miRNAs reduced Mcl-1 protein expression, only ten of them targeted Mcl-1 through direct binding to the Mcl-1 3′ untranslated regions (3′-UTR) of the gene. Other resistance pathways and regulators of Mcl-1 expression may be identified using this method. Our data can be used to design miRNA replacement therapies to increase sensitivity to Bcl-2 antagonists. The expression of the 12 marker microRNAs is now being correlated to the sensitivity to ABT-263 in order to enable rational patient selection in ABT-263 clinical trials.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B4
Abstract B48: Expression of metalloproteinases mmp-2, mmp-9 in sentinel lymph node and sera of patients with breast cancer
Authors: Daniele Antonella , Quaranta Michele , Divella Rosa , Zito Alfredo Francesco , Giannelli Gianluigi , Asselti Maria Consiglia , Mazzocca Antonio , Paradiso Angelo
Published: January 2011

Background: A sentinel lymph node is the first lymph node to receive lymphatic drainage from a tumour. In patients with breast cancer, several studies suggest that sentinel lymph node biopsy can accurately predict the status of axillary lymph nodes and so avoid them removal. Invasion and metastasis are the most important problems in the management of breast cancer. These events require diverse proteolytic enzymes among which MMP-2 and MMP-9 play a significant role in degradation of type IV collagen, the major component of the Basement Membrane (BM). The main objective of this study is to evaluate the expression of MMP-2 and MMP-9 in sentinel lymph node and serum of breast cancer patients at the aim to evaluate their clinical usefulness as diagnostic tumour markers in breast cancer.

Material and Methods: Expression of MMP-2 and MMP-9 was performed on sentinel lymph node by immunohistochemistry while gelatine zymography was used to determinate the serum expression of both Metalloproteinases. The association of MMP-2 and MMP-9 with clinicopathological features, including histological type, estrogen (ER) and progesterone (PgR), cancer familiarity and CA 15.3 levels were also analysed. In all 50 metastatic e non metastatic breast cancer patients and 34 healthy women were involved.

Results: MMP-2 and MMP-9 expression were statistically significant higher in metastatic breast cancer patients respect to non metastatic cancer patients and control group both in the sentinel lymph node and serum (p< 0.01). Besides our results show a statistically significant correlation between higher of MMP-2 and MMP-9 and cancer familiarity (p <0.01; p< 0.05) and between MMP-9 and CA 15.3 levels and MMP-9 and grade (G3).

Conclusion: Our date suggest a possible significant clinical utility of these proteolytic markers in malignant tumour, growth, invasion and metastasis in breast cancer and the role of sentinel lymph node to predict a possible involvement of axillary lymph nodes.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B48
Abstract B7: The bone metastasis secretome: Identification of potential therapeutic targets via high-throughput proteomics
Authors: Blanco Mario Andres , LeRoy Gary , Zee Barry , Aleckovic Maša , Garcia Benjamin , Kang Yibin
Published: January 2011

Secreted tumor proteins are critical for establishing the requisite tumor-stromal interactions in the process of metastasis and are superior therapeutic targets. Here we present a proteome-scale analysis to identify novel secreted proteins associated with bone metastasis. To characterize the complete set of secreted proteins involved in tissue-specific metastasis, we analyzed the secreted protein fractions from four sublines of the MDA-MB-231 breast cancer cell line which differ in bone metastasis ability. Using LC-MS/MS mass spectrometry technology, we identified 1153 total unique proteins from these fractions and filtered out those proteins predicted to be non-secreted using SignalP 3.0. This analysis yielded 206 unique secretory-predicted proteins, thus providing the first view of the MDA-MB-231 secretome. To determine potential therapeutic targets, we looked at which secreted proteins were found only in the highly bone metastatic cell lines. This 32-protein bone metastasis “secretome signature” contained several proteins already known to play a role in promoting bone metastasis (eg. MMP1, CTGF, and TGF-) as well as many novel proteins with no prior link to metastasis or cancer. GO categories significantly enriched in this signature included “extracellular region,” “calcium ion binding,” and “ IGF-binding,” further confirming its biological relevance. Oncomine database analysis indicated that 6 of 32 secretome signature proteins are strongly upregulated in multiple human cancers as compared to matched normal tissue. To narrow the secretome signature biologically, we repeated the LC-MS/MS analysis on two additional sets of matched parental vs. highly bone metastatic derivative cell lines. From the three total secretome analyses, 5 secreted proteins were found to be unique to bone metastatic derivatives in two of three cell types, and one protein was found in every bone metastatic derivative (and in none of the weakly bone metastatic lines). Oncomine analysis indicated that three of these five proteins were also strongly upregulated in multiple human cancers as compared to normal tissue. This analysis of the metastasis secretome and its associated bone metastasis-specific changes has identified multiple novel secreted proteins strongly implicated in clinical malignant progression. Following experimental and clinical validation, any could be considered for therapeutic targeting via monoclonal antibody.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B7
Abstract B9: Germline conservation of hypermutable codons in gatekeeper tumor suppressor genes implicates pro-carcinogenic CpG sites in vivo as anti-teratogenic ‘shock absorbers’ in utero
Authors: Zhao Yongzhong , Epstein Richard J.
Published: January 2011

Purpose: Methylated CpG sites are readily mutated, yet are strongly conserved in tumor suppressor genes. Uniquely, the arginine-encoding CGA codon may undergo methylation-dependent point mutation to a stop codon (TGA), whereas the arginine-encoding AGA codon is stable. To clarify the relationship between germline codon mutability and somatic carcinogenesis, we compared patterns of codon usage in tumor suppressor gene subsets known to differ in function and evolutionary stability.

Procedures: Human tumor suppressor genes were classified as either pro-apoptotic (gatekeepers; high-essentiality, low Ka/Ks) or repair-type (caretakers; low-essentiality, high Ka/Ks). Reference normal and tumor gene sequence databases were analysed in terms of nucleotide composition (GC%), intragenic CpG sites, and CGA/AGA codon patterns. Phylogenetic analyses of relevant codon frequencies across 24 species from the UCSC database were performed as a control.

Results: Species-specific stop codon frequency varies with genomic GC content in a methylation-regulated manner, whereas CGA frequency is GC-independent. Whole-genome frequencies of CGA and TGA codons are positively correlated (p = 0.013). CGA mutation in tumors is asymmetric (TGA >> CAA; p < 0.001), with such mutations 3-fold more likely to be nonsense-type in gatekeepers than in caretakers. Non-random CGA clustering is demonstrable in canonical gatekeepers such as NF1, Rb and TP53 (p < 0.02).

Conclusion: Human pro-apoptotic suppressor genes are selectively enriched for the presence of nonsense-prone CpG-containing codons involved in somatic adult-onset tumor progression. We propose that this germline vulnerability is conserved as part of a methylation-dependent embryonic response to environmental teratogens which otherwise cause excessive apoptosis, somatic malformations, and reduced fitness.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B9
Abstract CN2-2: Development of therapeutic cancer vaccine and construction of clinical research network in Japan
Authors: Nakamura Yusuke
Published: January 2011

cDNA microarray technologies have enabled us to obtain comprehensive data for gene expression profiles of human cancers. To isolate novel molecular targets for diagnosis and treatment of cancer, we constructed expression profile database of cancer cells originated from various organs with their corresponding non-cancerous tissues using a cDNA microarray that consists of more than 30,000 genes or ESTs. These experiments disclosed a number of genes that appeared to be involved in development and/or progression of cancers in those tissues. We also constructed expression profile database for 30 normal human tissues/organs. So far, we have analyzed approximately 1,400 clinical cancer samples of the liver, pancreas, stomach, colon, esophagus, bile duct, uterus, lung, ovary, kidney, urinary bladder, testis, prostate, breast, and soft tissues as well as acute and chronic myeloid leukemias. We have selected hundreds of candidate genes by the following criteria; (1) gene expressions were transactivated in a large proportion of cancer tissues in comparison with their corresponding normal tissues and (2) expression was not observed or hardly detectable in any of important vital organs. The further functional analysis of these molecules identified dozens of genes that are likely to function as oncogenes in various cancers. The suppression of expression of such genes with siRNA induced cell cycle arrest, apoptosis, or suppression of anchoring-dependent cell growth. We screened 9- or 10-amino-acid peptides corresponding to a part of such oncoantigens that induce cytotoxic T lymphocytes that would specifically kill cancer cells in an HLA-A restricted and antigen-dependent manner. We have already isolated nearly 60 peptides (HLA-A02 or HLA-A24 restricted) derived from about 50 oncoantigens and started translational research using some of them in August 2006. We have organized a group that performs translational research and now consists of ∼60 hospitals (Cancer Peptide Vaccine Translational Research Network = Captivation Network). We are now running more than 30 different protocols and more than 900 cancer patients have been enrolled by the end of 2009. The promising clinical data and immunological analysis of our translational research will be introduced in the meeting. These results indicated that systematic expression analysis should be a very effective approach for identification of molecules that are potential targets for development of novel therapeutic drugs.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN2-2
Abstract ED1-1: The role of informatics in translational research: The interplay of epidemiology, clinical studies, and discovery research
Authors: Koehler Jacob
Published: January 2011

Whilst drug discovery costs are increasing, the number of successful new drugs is decreasing. Lack of efficacy, toxicology and commercial reasons are the main reasons why drugs are discontinued. Further, there is an ever growing demand to demonstrate safety and comparative effectiveness to payers, regulatory agencies and patients. A range of computational, data driven translational approaches provide new opportunities for improving drug discovery, development and utilization.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):ED1-1
Abstract ED4-1: Functional imaging: Beyond FDG
Authors: Price Patricia M.
Published: January 2011

Whole body FDG has become an important part of the diagnosis and staging of cancer. Worldwide, over 90% of PET imaging is now clinical whole body FDG imaging. However, in vivo molecular imaging with PET is an emerging discipline that is of importance to the development of anti-cancer therapies. As anti-cancer strategies become more directed towards a defined molecular target, we need information that is relevant to humans about whether the molecular target is expressed, the selectivity and binding of the compound for that target, and the effects of such an interaction.

PET is the most sensitive and specific technique for imaging molecular pathways in vivo in humans. PET uses positron emitting radionuclides to label molecules which can then be imaged in vivo. The inherent sensitivity and specificity of PET is the major strength of this technique, able to provide quantitative kinetic information down to sub-picomolar levels.

Molecular imaging with PET can provide pharmacokinetic, pharmacodynamic and mechanistic information. Use of the technique in early clinical trials can (1) provide information on optimum biological dose and PK/PD relationships; (2) identify tumours containing specific molecular targets; and (3) provide in vivo pharmacodynamic evaluation of compounds.

Molecular imaging with PET has the potential to ensure a paradigm shift in drug development, to revolutionise, streamline and accelerate drug development in oncology. Examples will presented where in vivo clinical tumour and normal tissue pharmacokinetics measured by PET have helped drug development, from use with small molecules to antisense therapy.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):ED4-1
Abstract KN1: Finding mechanisms and biomarkers of drug resistance in cancer
Authors: Bernards René
Published: January 2011

Unresponsiveness to therapy remains a significant problem in the treatment of cancer, also with the new classes of targeted therapeutics. In my laboratory, we use functional genetic approaches to identify biomarkers that can predict responsiveness to clinically relevant cancer therapeutics. We focus on targeted cancer drugs such as trastuzumab (Herceptin), PI3K inhibitors, MEK inhibitors, ALK inhibitors, mTOR inhibitors, Histone Deacetylase inhibitors and retinoic acid. These drug target specific molecules or pathways that are often activated in cancer. Nevertheless, it remains poorly explained why a significant number of tumors do not respond to the therapy. We aim to elucidate the molecular pathways that contribute to unresponsiveness to targeted cancer therapeutics using a functional genetic approach. This will yield biomarkers that may be useful to predict how individual patients will respond to these drugs. Furthermore, this work may allow the development of drugs that act in synergy with the established drug in the treatment of cancer. To identify biomarkers that control tumor cell responsiveness to cancer therapeutics, we use two complementary approaches. First, we use genome wide loss-of-function genetic screens (with shRNA interference libraries) in cancer cells that are sensitive to the drug-of-interest to search for genes whose down-regulation confers resistance to the drug-of-interest (resistance screens). In addition, we use single well siRNA screens with a low dose of the drug to screen for genes whose inhibition enhances the toxicity of the cancer drug (sensitizer screens). Once we have identified resistance and/or sensitizer genes, we ask if their expression is correlated with clinical response to the drug-of-interest using tumor samples of cancer patients treated with the drug in question, whose response to therapy is documented. Examples of genetic screens to identify mechanisms of resistance to different cancer drugs will be presented.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):KN1
Abstract PL3-2: Translating multiple myeloma stem cells
Authors: Matsui William
Published: January 2011

Emerging evidence suggest that many human cancers consist of phenotypically and functionally heterogeneous cell types. Moreover, the capacity for clonogenic growth and self-renewal may be restricted to specific cell populations. In multiple myeloma, malignant plasma cells make up the vast majority of tumor cells and are responsible for symptoms arising within the disease. However, myeloma plasma cells also appear to be terminally differentiated like their normal counterparts and incapable of long-term proliferation. Similar to other laboratories, we have identified minor populations of B cells that share the identical immunoglobulin gene rearrangements as the malignant plasma cells in most myeloma patients. We have further studied the functional capacity of these clonotypic B cells and found that they have ability to produce ectopic tumor growth both in vitro and in immunodeficient mice in which they recapitulate human disease. Moreover, these cells are relatively resistant to several agents currently used to treat multiple myeloma suggesting that they persist following treatment and are responsible for tumor regrowth and disease relapse. In order to inhibit these drug resistant cancer stem cells, we have studied cellular pathways that regulate the self-renewal of normal adult stem cells and found that several, including the developmental pathway Hedgehog, are potential therapeutic targets. Lastly, we have begun to develop biomarker strategies to serially quantify myeloma stem cells in patients undergoing treatment. We hope that these will serve as useful endpoints as myeloma stem cell directed therapies enter the clinic.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):PL3-2
Abstract PL4-1: Immunotherapy of established lesions caused by high-risk HPV
Authors: Melief C.J.M. , Welters M.J.P. , Valentijn A.R.P.M. , Berends-van der Meer T.M.A. , Löwik M.J.G. , Vloon A.P.G. , Essahsah F. , Fathers L.M. , Offringa R. , Drijfhout J.W. , Oostendorp J. , Fleuren G.J. , van der Burg S.H. , Kenter G.G.
Published: January 2011

Therapeutic vaccination with a synthetic long peptide (SLP®) vaccine mediated the eradication of established human papilloma virus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papilloma virus. Subsequent phase I/II studies with an HPV16 SLP® vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. The purpose of the current study was to test the clinical efficacy of this HPV16 SLP® vaccine in HPV16-induced high grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high.

In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses, determined before and after vaccination.

The vaccine was safe, as no side effects exceeding CTC grade 2 were observed. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions as compared to non-responders.

This study shows that in women with VIN3 objective clinical responses can be achieved by therapeutic vaccination with synthetic long peptides that is able to induce effective HVP16-specific T-cell responses.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):PL4-1
Abstract PL6-1: Resistance to targeted therapies in a realistic mouse breast cancer model
Authors: Borst Piet , Rottenberg Sven , Jonkers Jos , Zander Serge , Pajic Marina , Jaspers Janneke
Published: January 2011

The contribution of ABC-transporters to resistance in patients has remained controversial. We have tackled this problem in a mammary mouse tumor model. These Brca1−/−, p53−/− tumors arise “spontaneously” and closely resemble human breast cancer in BRCA1+/− carriers (Rottenberg, PNAS 2007;104:12117–22; 2008;105:17079–84). The tumors respond to chemotherapeutic agents used in human breast cancer, but eventually end up being resistant to most drugs.

Resistance to doxorubicin in this tumor model is nearly always due to P-glycoprotein upregulation at the transcriptional level. A 5-fold increase in the low basal level of P-glycoprotein RNA in these tumors is sufficient for complete resistance. These levels of P-glycoprotein are not detectable with standard immunocytochemistry using the C219 Mab (Pajic, Cancer Res 2009;69:6396–404). Crossing in null alleles for Mdr1a/b made the tumors hypersensitive to doxorubicin, docetaxel, and to the PARP inhibitor olaparib. In about half of the mice treated with topotecan, resistance is associated with upregulation of Abcg2 (Bcrp). Tumors in Abcg2−/− mice take longer to become resistant (Zander, Cancer Res 2009 in press). Notwithstanding the complete remissions often obtained with some drugs, we are rarely able to eradicate the tumor. This is not due to the hypothetical unique properties of tumor stem cells. Supported by Dutch Cancer Foundation and EU Framework Program.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):PL6-1
Abstract PL6-2: Antibody-initiated cancer immunotherapy
Authors: Weiner Louis M. , Tang Yong , Wang Shangzi , Surana Rishi , Xu Wei , Zhou Yu , Marks James D. , Golemis Erica
Published: January 2011

We have previously described the critical structural determinants of anti-tumor antibodies that promote in vivo tumor targeting and antibody-dependent cellular cytotoxicity (ADCC). We now describe treatment strategies in murine models that 1) improve in vitro and in vivo ADCC, 2) dissect the relative contributions of tumor signaling perturbation and ADCC for in vivo therapeutic efficacy, and 3) identify therapy conditions that promote the induction of host-protective adaptive immunity. We also have identified tumor-intrinsic factors that sensitize tumor cells to EGFR-directed antibody therapy, using a siRNA library focused on genes functionally linked to the EGFR signaling pathway. We predict that these findings will be important for the improved efficacy of EGFR family-directed antibody therapies. Supported by CA50633, CA121033.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):PL6-2
Preclinical Studies on the Mechanism of Action and the Anti-Lymphoma Activity of the Novel Anti-CD20 Antibody GA101
Authors: Dalle Stephane , Reslan Lina , Besseyre de Horts Timothee , Herveau Stephanie , Herting Frank , Plesa Adriana , Friess Thomas , Umana Pablo , Klein Christian , Dumontet Charles
Published: January 2011

GA101 is a novel glycoengineered Type II CD20 monoclonal antibody. When compared with rituximab, it mediates less complement-dependent cytotoxicity (CDC). As expected for a Type II antibody, GA101 appears not to act through CDC and is more potent than the Type I antibody rituximab in inducing cell death via nonclassical induction of apoptosis cytotoxicity, with more direct cytotoxicity and more antibody-dependent cell-mediated cytotoxicity. We evaluated the antitumor activity of GA101 against the human-transformed follicular lymphoma RL model in vivo in severe combined immunodeficient mice (SCID) mice. GA101 induced stronger inhibition of tumor growth than rituximab. Combination of GA101 with cyclophosphamide in vivo confirmed the superiority of GA101 over rituximab. Neutralizing the complement system with cobra venom factor partially impaired the antitumor activity of rituximab, but had no impact on the efficacy of GA101. In vitro GA101 more potently induced cell death of RL cells than rituximab. The expression of a limited number of genes was found to be induced by both antibodies after exposure in vitro. Among these, early growth response 1 and activation transcription factor 3 were confirmed to be increased at the protein level, suggesting a possible role of these proteins in the apoptotic signalling of anti-CD20 antibodies. These data imply that GA101 is superior to rituximab not only as a single agent, but also in combination with chemotherapy. These data suggest the presence of novel signalization pathways activated after exposure to anti-CD20 antibodies. Mol Cancer Ther; 10(1); 178–85. ©2011 AACR.
Celecoxib Can Induce Vascular Endothelial Growth Factor Expression and Tumor Angiogenesis
Authors: Xu Kaiming , Gao Huiying , Shu Hui-Kuo G.
Published: January 2011

Increased COX-2 expression has been linked to increased angiogenesis and a worse prognosis in patients with malignant gliomas and other tumor types. This led to our interest in assessing the response of glioma cell lines to treatment with celecoxib, a selective COX-2 inhibitor. However, contrary to its reported antiangiogenic effects, treatment with celecoxib actually induced the expression of VEGF in multiple glioma as well as other cancer cell lines. This induction of VEGF was comparable to, if not greater than, that found after exposure of cells to hypoxia. Pharmacologic inhibition and siRNA silencing of p38-mitogen-activated protein kinase and the Sp1 transcription factor revealed their involvement in this celecoxib-induced VEGF expression. Consistent with the documented role of Sp1 in this effect, VEGF induction was found to involve transcriptional activation and not to change the stability of VEGF mRNA. The biological significance of this effect was confirmed in vivo by showing both induction of VEGF expression and microvessel density in tumor xenografts and increased angiogenesis in a matrigel plug assay in nude mice that were administered celecoxib. We speculate that treatment with celecoxib may, in some instances, enhance tumor cell expression of VEGF as well as angiogenesis and, consequently, may have detrimental effects on the response of tumors to this drug. Mol Cancer Ther; 10(1); 138–47. ©2011 AACR.
Drug Efflux by Breast Cancer Resistance Protein Is a Mechanism of Resistance to the Benzimidazole Insulin-Like Growth Factor Receptor/Insulin Receptor Inhibitor, BMS-536924
Authors: Hou Xiaonan , Huang Fei , Carboni Joan M. , Flatten Karen , Asmann Yan W. , Ten Eyck Cynthia , Nakanishi Takeo , Tibodeau Jennifer D. , Ross Douglas D. , Gottardis Marco M. , Erlichman Charles , Kaufmann Scott H. , Haluska Paul
Published: January 2011

Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism. Mol Cancer Ther; 10(1); 117–25. ©2011 AACR.
Lysophosphatidylcholine Pretreatment Reduces VLA-4 and P-Selectin–Mediated B16.F10 Melanoma Cell Adhesion In vitro and Inhibits Metastasis-Like Lung Invasion In vivo
Authors: Jantscheff Peter , Schlesinger Martin , Fritzsche Juliane , Taylor Lenka A. , Graeser Ralph , Kirfel Gregor , Fürst Dieter O. , Massing Ulrich , Bendas Gerd
Published: January 2011

Lysophosphatidylcholine (LysoPC) is an important intermediate in degradation and biosynthesis of phosphatidylcholine (PC). Reduced plasma LysoPC levels observed in patients with advanced cancer indicate a deregulation of LysoPC metabolism in metastasis. Recent data showed strong antimetastatic effects of liposomes consisting of saturated PC in a murine pancreatic metastasis model. LysoPC, generated from saturated PC after accumulation of the liposomes in tumor tissue, might be contributing to these effects. Examining effects of high local concentrations of saturated LysoPC and investigating potential molecular mechanisms, fast removal of saturated LysoPC from medium by murine B16.F10 melanoma cells and radical shifts in tumor cell membrane fatty acid (FA) composition toward saturated FAs were observed in vitro. Scanning electron microscopy revealed remarkable morphologic surface changes of LysoPC-treated tumor cells, probably causing their impaired migratory ability on fibronectin. A LysoPC concentration exceeding a threshold of about 400 μmol/L, slightly above physiologic levels, strongly reduced VLA-4–mediated binding of B16.F10 cells to VCAM-1 as well as P-selectin–dependent interaction with activated platelets, although expression levels were not altered. These findings were reflected in a syngenic intravenous lung invasion model using repeatedly ex vivo LysoPC-treated (450 μmol/L) B16.F10 cells, resulting in significantly reduced lung metastasis-like lesions (−48.3%, P = 0.006). Prior application of 50 IU unfractionated heparin further reduced lung invasion (−81.6%, P = 0.043). Our work shows for the first time that saturated LysoPC in high concentrations reduces melanoma cell adhesion in vitro and hematogeneous dissemination in vivo by direct ex vivo tumor cell targeting. Mol Cancer Ther; 10(1); 186–97. ©2011 AACR.
Prediction of Colorectal Cancer Relapse and Prognosis by Tissue mRNA Levels of NDRG2
Authors: Chu Dake , Zhang Zixi , Li Yunming , Wu Lin , Zhang Jing , Wang Weizhong , Zhang Jian
Published: January 2011

NDRG2 (N-Myc downstream-regulated gene 2) is aberrantly expressed in colorectal cancer (CRC) and related to tumor differentiation status. In the present study, we investigated the association between NDRG2 mRNA levels in primary CRC to determine whether levels of NDRG2 mRNA could predict relapse and survival. A hospital-based study cohort of 226 CRC patients was involved in the study. NDRG2 mRNA levels were determined by real-time PCR. Correlations of NDRG2 mRNA expression with tumor clinicopathologic features, disease-free survival, and overall survival of the patients were studied. Significant decreased expression of NDRG2 mRNA was detected in tumor specimens. NDRG2 mRNA expression significantly correlated with differentiation status (P < 0.001), lymph node metastasis (P < 0.001), and tumor node metastasis stage (P < 0.001). Patients with reduced level of NDRG2 mRNA had a statistically significantly shorter disease-free survival and overall survival duration than patients with preserved expression of NDRG2 mRNA. In multivariate analysis, NDRG2 mRNA level was found to be an independent prognostic factor for both disease-free survival and overall survival of CRC patients. The present research provided the first evidence that decreased NDRG2 mRNA expression in primary human CRC might be a powerful, independent predictor of recurrence and outcome. Mol Cancer Ther; 10(1); 47–56. ©2011 AACR.
MEK/ERK Inhibitor U0126 Increases the Radiosensitivity of Rhabdomyosarcoma Cells In vitro and In vivo by Downregulating Growth and DNA Repair Signals
Authors: Marampon Francesco , Gravina Giovanni Luca , Di Rocco Agnese , Bonfili Pierluigi , Di Staso Mario , Fardella Caterina , Polidoro Lorella , Ciccarelli Carmela , Festuccia Claudio , Popov Vladimir M. , Pestell Richard G. , Tombolini Vincenzo , Zani Bianca Maria
Published: January 2011

Multimodal treatment has improved the outcome of many solid tumors, and in some cases the use of radiosensitizers has significantly contributed to this gain. Activation of the extracellular signaling kinase pathway (MEK/ERK) generally results in stimulation of cell growth and confers a survival advantage playing the major role in human cancer. The potential involvement of this pathway in cellular radiosensitivity remains unclear. We previously reported that the disruption of c-Myc through MEK/ERK inhibition blocks the expression of the transformed phenotype; affects in vitro and in vivo growth and angiogenic signaling; and induces myogenic differentiation in the embryonal rhabdomyosarcoma (ERMS) cell lines (RD). This study was designed to examine whether the ERK pathway affects intrinsic radiosensitivity of rhabdomyosarcoma cancer cells. Exponentially growing human ERMS, RD, xenograft-derived RD-M1, and TE671 cell lines were used. The specific MEK/ERK inhibitor, U0126, reduced the clonogenic potential of the three cell lines, and was affected by radiation. U0126 inhibited phospho-active ERK1/2 and reduced DNA protein kinase catalytic subunit (DNA-PKcs) suggesting that ERKs and DNA-PKcs cooperate in radioprotection of rhabdomyosarcoma cells. The TE671 cell line xenotransplanted in mice showed a reduction in tumor mass and increase in the time of tumor progression with U0126 treatment associated with reduced DNA-PKcs, an effect enhanced by radiotherapy. Thus, our results show that MEK/ERK inhibition enhances radiosensitivity of rhabdomyosarcoma cells suggesting a rational approach in combination with radiotherapy. Mol Cancer Ther; 10(1); 159–68. ©2011 AACR.
Targeting Tyrosine Phosphorylation of PCNA Inhibits Prostate Cancer Growth
Authors: Zhao Huajun , Lo Yuan-Hung , Ma Li , Waltz Susan E. , Gray Jerilyn K. , Hung Mien-Chie , Wang Shao-Chun
Published: January 2011

The proliferation cell nuclear antigen (PCNA) is a critical protein required for DNA replication in proliferating cells including cancer cells. However, direct inhibition of PCNA in cancer cells has been difficult due to the lack of targetable sites. We previously reported that phosphorylation of tyrosine 211 (Y211) on PCNA is important for the proliferative function of PCNA when this protein is associated with the chromatin in cancer cells. Here, we show that the Y211 phosphorylation of PCNA is a frequent event in advanced prostate cancer. To explore the potential of this signaling event in inhibition of cancer cell growth, we used a synthetic peptide, the Y211F peptide, which when present inhibits phosphorylation of Y211 on endogenous PCNA. Treatment with this peptide, but not a scrambled control peptide, resulted in S-phase arrest, inhibition of DNA synthesis, and enhanced cell death in a panel of human prostate cancer cell lines. In addition, treatment with the Y211F peptide led to decreased tumor growth in PC3-derived xenograft tumors in vivo in nude mice. Our study shows for the first time that PCNA phosphorylation at Y211 is a frequent and biologically important signaling event in prostate cancer. This study also shows a proof of concept that Y211 phosphorylation of PCNA may be used as a therapeutic target in prostate cancer cells, including cells of advanced cancers that are refractory to standard hormonal therapies. Mol Cancer Ther; 10(1); 29–36. ©2011 AACR.
Abstract A100: STAT3 as a novel cancer prevention target in colorectal cancer stem cells
Authors: Lin Li , Li Chenglong , Li Pui-Kai , Fuchs James , Lin Jiayuh
Published: January 2011

Constitutive activation of signal transducers and activators of transcription 3 (STAT3) signaling is frequently detected in human cancer including colon cancer, and has emerged as an attractive molecular target for cancer prevention. Recent experimental evidence suggests that the existence of a small population of tumorigenic stem/progenitor cells may be responsible for tumor initiation, chemotherapy and radiation resistance, invasion, recurrence, and metastasis. An increasing body of evidence suggests that the cancer stem cell concept is also relevant to colorectal cancer. To date, however, whether STAT3 is activated in colon cancer stem cells or cancer-initiating cells and what the role of STAT3 signaling may play in these cancer stem cells is still unknown. If STAT3 is activated in colorectal cancer stem cells, inhibiting STAT3 may offer a promising opportunity to target colorectal cancer stem cells and prevent the recurrence of cancer. We utilized the colon cancer stem cells, which are characterized by an aldehyde dehydrogenase (ALDH)-positive (ALDH+) and CD133-positive (CD133+) subpopulation. We demonstrated that ALDH+/CD133+ cells have capacity than ALDH-/CD133- cells to form more tumorspheres and to exhibit more potent tumor-initiating ability in mice. We then examined the STAT3 activation in these colon cancer cells. Interestingly, we observed that the ALDH+/CD133+ subpopulation of colon cancer cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, compared to the ALDH-/CD133- subpopulation and un-separated colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. We demonstrated that dietary agent, curcumin and a new curcumin analog, GO-Y030 can inhibit STAT3 phosphorylation, cell viability, and induced apoptosis in colon cancer stem cells. The inhibition of STAT3 was confirmed using a novel STAT3-selecitve curcumin analog, FLLL32, STAT3 ShRNA, as well as STAT3 inhibitors, LLL12 and Stattic. All these STAT3 inhibitors, inhibited STAT3 phosphorylation, cell viability, and tumorsphere growth in colon cancer stem cells. Furthermore, both GO-Y030 and LLL12 inhibited tumor growth of colon cancer stem cells in NOD/SCID mouse model in vivo. In Summary, this is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in colon cancer stem cells. Our study is also the first attempt to target STAT3 in colon cancer stem cells and we demonstrated for the first time that colon cancer stem cells are indeed sensitive to the inhibition by small molecular STAT3 inhibitors, FLLL32, LLL12, Stattic, STAT3 shRNA, and curcumin analog, GO-Y030. Our results suggest that STAT3 is a novel prevention target in colon cancer stem cells and inhibition of activated STAT3 in cancer stem cells may offer an effective preventive approach for colorectal carcinoma.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A100.
Abstract A102: Chemopreventive efficacy and mechanistic studies of Targretin in the B[a]P-induced mouse lung tumor prevention model
Authors: Sharma Sheela , Lee Jin , Zhou Jianliang , Steele Vernon E.
Published: January 2011

Aspiration route has been demonstrated to be as effective as inhalation in administering agents to lung with associated reduction in cost for equipment and chemopreventive agent. The current study evaluated the chemopreventive efficacy and mechanism (s) of Targretin, a novel oral synthetic retinoid that selectively binds and activates retinoid X receptors, via oropharyngeal aspiration against the strain A/J mouse lung adenoma induced by benzo[a]pyrene (B[a]P). Lung adenomas were induced in 8-week old female strain A/J mice by three oral gavages of B[a]P (2 mg /0.2 ml cotton seed oil). The mice were allowed 4 weeks to develop dysplasia, then given one of three doses of Targretin (0.1, 0.3 and 1 mg/kg) for 16 weeks. At different time points, the expression of a series of biomarkers (e.g., Cox-2 and 5-Lox at 2 and 16 weeks, survivin, PCNA and apoptosis at 16 weeks) in lung was evaluated by Western Blot and immunohistochemistry. Tumor incidence and multiplicity in the lung were measured following 16-week aspiration of Targretin and compared with B[a] P only group. Targretin showed dose-related inhibition of B[a]P-induced tumor incidence (p<0.05) at 0.1 and 0.3 mg/kg following 16-week treatment with no effect on tumor multiplicity. Targretin also showed dose-dependent inhibition of 5-Lox (25-47%) at 2 and 16 weeks, and Cox-2 (27-38%) and survivin (39-48%) at 16 weeks. A dose-dependent induction of apoptosis (2.2-3.0-fold) and a marginal but dose-dependent inhibition of PCNA were also observed at 16 weeks. In conclusion, this study demonstrated that Targretin via aspiration showed chemopreventive efficacy against mouse lung adenoma with good correlation to early and late biomarkers of lung cancer progression. Also, it indicated that the aspiration route can be an excellent inexpensive alternative to inhalation for direct drug delivery to the rodent lung for efficacy testing of potential chemopreventive agents (Supported by NCI #N01-CN-53301).

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A102.
Abstract A107: Epigenetic markers for tissue-based breast cancer risk stratification
Authors: Euhus David M. , Bu Dawei , Rao Roshni , Leitch Marilyn , Lewis Cheryl
Published: January 2011

Effective breast cancer risk management requires accurate breast cancer risk stratification. The Gail model is the most widely used and best validated epidemiological risk assessment model. Though well calibrated, this model is not ideal for individualized risk counseling as it is only 8% better than chance for discriminating between women with breast cancer and unaffected women. Biologically based approaches, such as cytological assessment of nipple aspirate fluid or assessment of mammographic density, only marginally improve the discrimination of the Gail model. Promoter region methylation is one of the earliest identifiable molecular changes during breast carcinogenesis. We have previously shown that RASSF1A methylation, detected in benign breast epithelium obtained by random periareolar fine needle aspiration biopsy (RP-FNA), is associated with increased breast cancer risk, but this was based primarily on correlation with Gail risk and not cancer case prediction. A clinically useful epigenetic risk stratification panel would be ideally suited to benign RP-FNA samples and would provide a high degree of discrimination between benign samples from women with cancer and unaffected women. We performed a genome-wide 5-aza-2’-deoxycytidine (5-aza) induced gene re-expression screen in 6 breast cancer cell lines and 6 early passage primary benign breast epithelial cell cultures to identify potential epigenetic markers of breast cancer risk. MSP assays were designed for the 286 genes that were 1) expressed in benign breast epithelium, 2) not expressed in cancer, and 3) induced by 5-aza in cancer, or 4) had previously shown some potential for breast cancer risk stratification. 20 genes were identified that were frequently methylated in primary breast cancer, rarely methylated in benign RP-FNA samples and never methylated in lymphocytes. Quantitative multiplex methylation-specific PCR assays were designed and optimized for the 17 best genes and then assessed in an archival RP-FNA sample set that included 146 samples from unaffected women, 59 benign samples from breast cancer patients, and 52 primary breast cancers. A three marker panel including PSAT1, HS3ST2, and GNE provided the best discrimination between benign samples from cancer patients and unaffected women. One or more of these markers was scored as positive in 46% of benign samples from cancer patients, 8% of samples from unaffected patients, and 72% of primary cancers. The odds ratio for cancer case prediction based on assessment of benign RP-FNA samples was 9.42 (95% CI 4.31 — 20.59, p < 0.0001). These observations will be validated in an independent set of 160 sample prospectively acquired for this purpose. These data suggest that a limited epigenetic marker panel can detect a small population of breast cells accessible by RP-FNA whose presence is associated with significantly increased breast cancer risk.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A107.