bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B48: Germline mutations in BRCA1 and BRCA2 as predictive biomarkers of therapy response
Authors: Tassotto Mary Lynn , Sutherland Lindsay , Th'ng John
Published: May 2012

The results of in vitro chemosensitivity studies have shown some consistency with response to chemotherapy in patients, indicating that this approach may be used to identify patients that are resistant to conventional chemotherapy regimens. However, this approach is not routinely used in an adjuvant or neoadjuvant setting as a treatment strategy. Treatment selection for breast and ovarian cancer is currently based on pathological classification of the tumour, without assessment of in vitro chemosensitivity. BRCA1 and BRCA2 germline mutations are considered to be both prognostic cancer biomarkers and predictive biomarkers of breast and ovarian cancer susceptibility; however, their utilization as predictive biomarkers of therapy response has not been investigated. Expression levels of BRCA1 modulate chemosensitivity in breast cancer cell lines, and epigenetic regulation of BRCA1 leading to decreased BRCA1 expression is often observed in sporadic breast cancer. We have hypothesized that cells with mutations in BRCA1 or BRCA2 may be differentially sensitive to DNA damaging agents used in cancer therapy due to decreased levels of BRCA1 or BRCA2 gene expression in these cells. Using lymphocytes isolated from individuals with germline mutations in BRCA1 and BRCA2, we will present results showing the in vitro response to ionizing radiation and chemosensitivity to Paclitaxel and Doxorubicin, two chemotherapeutic agents commonly used in the adjuvant systemic treatment of breast cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):B48.
Abstract B54: Identification of the PI3K-mTOR pathway as regulator of mutant p53 turnover in human cancer cells
Authors: Bug Monika , Dobbelstein Matthias
Published: May 2012

The tumor suppressor p53 plays a crucial role in our cells to induce cell cycle arrest or apoptosis in response to different kinds of genotoxic stresses. In 50% of all solid tumors, p53 is mutated by a single point mutation within the DNA-binding domain and thereby loses its activity as a tumor suppressor. However, at the same time, mutant p53 actively contributes to chemoresistance and tumor progression through a gain of function (Dittmer et al., 1993). It was previously shown that mutant p53 highly accumulates in tumors of mice with targeted germ line mutations of the p53 gene (knock-in), whereas normal cells of these mice express it at low levels (Lang et al., 2004; Terzian et al., 2008). This argues that the deregulation of tumor-specific signaling cascades leads to an accumulation of mutant p53 and subsequently to tumor progression.

Our study aims at identifying these signaling pathways that contribute to the regulation of mutant p53 expression, and to describe their mechanisms. We performed quantitative analysis of mutant p53 protein levels in a cancer cell line after the individual knock-down of all known human kinases using automated microscopy (high content cell screening).

The bladder carcinoma derived cell line 5637 (carrying the p53 R280T mutation) was transfected with three different synthetic siRNAs targeting each known kinase or kinase regulator in a 96-well format, and p53 was detected by automated immunofluorescence microscopy. In each of the resulting 2400 cell populations the p53 expression levels were determined for at least 500 individual cells. Alterations in the distribution of p53 staining intensities were assessed for each kinase relative to non-targeting control siRNAs. The results of the kinases, that lead to the strongest up- or down-regulations of mutant p53 in the screen, were confirmed in different cell lines (U251 and U373, both derived from glioma, with the p53 hot spot mutation R273H), by immunofluorescence and immunoblot analysis.

We identified a group of kinases currently known to act in the PI3-kinase → mTOR pathway as potential regulators of mutant p53. The knock-down of these kinases resulted in a decrease of mutant p53 expression. Studying the mechanism of mutant p53 regulation, we performed quantitative real-time PCR after siRNA transfection and found no significant impairment of the p53 transcription. On the contrary, posttranslational modifications (phosphorylation and acetylation) of mutant p53 were decreased after knock-down of the identified kinases, even more strongly than the overall levels of the protein. These observations support the idea that the degradation of mutant p53 is decreased in cancer cells through alterations of PI3K-mTOR pathway associated kinases, resulting in p53 accumulation and oncogenic gain of function.

Citation Information: Cancer Res 2009;69(23 Suppl):B54.
Abstract B56: Identification of genes involved in mammary tumor susceptibility and resistance in two strains of mice
Authors: Griner Nicholas B. , Compton Shannon L. , Dickinson Ellen S. , Boehringer Michael , Wiesmüller Lisa , Jerry D. Joseph
Published: May 2012

Loss of p53 can lead to a variety of cancers, including breast cancer. Mice heterozygous for the p53 gene (designated Trp53+/−) develop spontaneous mammary tumors, but this depends on the strain background and has been linked to a locus on chromosome 7 (designated SuprMam1). Mammary tumors are common in BALB/c-Trp53+/−females, but are rare in C57BL/6-Trp53+/− mice. Prevalence of genomic instability appears to contribute to the phenotype as loss of heterozygosity (LOH) through mitotic recombination is significantly more common among tumors arising in BALB/c-Trp53+/− mice compared to C57BL/6J-Trp53+/− mice. This increased LOH in BALB/c-Trp53+/− tumors was shown to be due to recombination events. The Balb/c strain has been shown to have a deficiency in non-homologous end joining (NHEJ) of DNA double strand breaks (dsb), however, this does not account for the increase of LOH events in tumors. Our hypothesis is that BALB/c-Trp53+/− mice are more susceptible to mammary tumors due to impaired dsb repair leading to LOH. Using the COMET assay, we demonstrate that dsbs persist longer in BALB/c-Trp53+/− mouse embryonic fibroblasts (MEFs) compared to C57BL/6J-Trp53+/− MEFs. Similarly, co-localization of H2AX and the homologous recombination protein RAD51 remain at dsb longer in BALB/c-Trp53+/− MEFs compared to C57BL/6-Trp53+/− MEFs suggesting a decreased efficiency of homologous recombination. Using a GFP reporter assay, homology-directed repair was decreased by 36% in MEFs from BALB/c-Trp53+/− compared to C57BL/6-Trp53+/−. Palb2 is a candidate gene as it lies within the SuprMam1 interval and has been shown to contribute to heritable breast cancer. Although no coding SNPs were found within Palb2, preliminary results show the BALB/c-Trp53+/− cells have a 30% decrease in Palb2 RNA compared to C57BL/6-Trp53+/− cells as assayed by RT-qPCR. These results identify defects in homology-directed repair of dsb in BALB/c which are linked to susceptibility to mammary tumors in this strain. Experiments are in progress analyzing these features in congenic strains. The results will elucidate potential low penetrance modifiers that determine resistance to mammary tumors in both mice and humans which can be used as targets for therapy as well as breast cancer screening.

Citation Information: Cancer Res 2009;69(23 Suppl):B56.
Abstract B57: Regulation of Eag expression by p53 and Rb in tumoral cells
Authors: Chavez-Lopez Maria de Guadalupe , Garcia-Villa Enrique , Gariglio Patricio , Garrido E. , Hernandez-Gallegos Elizabeth , Ponce Veronica , Camacho Javier
Published: May 2012

Introduction: Ether à go-go (Eag) potassium channels display oncogenic properties. Their expression in healthy tissues is limited almost exclusively to the brain; however, it is highly expressed in several types of cancer. In addition, the inhibition of its activity or expression remarkably diminishes cellular proliferation and tumor progression. Our group found the expression of Eag in cells having alterations in p53 and Rb including C-33 and HeLa from cervical cancer. On the other hand, we have observed that Eag gene expression is regulated by factors that inhibit Rb or p53, such as, E6 and E7 oncogenes of the human papilloma virus and estradiol. Therefore, we have considered p53 and Rb like possible pathways for Eag regulation. To our knowledge, there are no reports on Eag regulation by tumor suppressor proteins. Thus in this work we evaluated the effect of p53 and Rb overexpression on the expression of Eag in C-33, HeLa (cervix) and A549 (lung cancer) cells. We also analyzed the expression of Eag in tumor samples from patients with retinoblastoma (a malignant tumor characterized by the loss of the gene Rb and alterations in the p53 pathway).

Methods: C-33, HeLa and A549 cells were obtained from American Type Culture Collection (ATCC) and the RNA's of patients with retinoblastoma from the Hospital of Pediatrics of the Medical Centre XXI century. In order to evaluate the expression of Eag, retrotranscription was performed and the analysis of gene expression was carried out by real time PCR, the results were analyzed by the method of ΔΔ-CT. In order to evaluate the effect of p53 and Rb overexpression on Eag, HeLa and C-33 cells were grown in DMEM and A549 cells on F12-K medium, supplemented with 10% of bovine fetal serum; later they were transfected with 1 µg of plasmid containing p53 and/or Rb. The RNA was obtained 72 hours later and then subjected to retrotranscription. Eag gene expression was then analyzed by real time PCR.

Results: We found that p53 and Rb transfection in C-33 cells does not affect the expression of Eag. However, Eag expression in HeLa cells was clearly down-regulated when transfected with p53 and/or Rb. Transfection of p53 in A549 cells diminished the expression of Eag, in contrast to Rb transfection which increased expression. Interestingly, we observed a very high expression of Eag in samples of retinoblastoma in comparison with the Eag expression in HeLa cells and in normal retina.

Conclusions: Our results suggest that tumor suppressor proteins p53 and Rb regulate the expression of Eag. In addition, our results also suggest Eag as a retinoblastoma potential therapeutic target for this type of tumors.

This project was partially supported by ICyT DF.

Citation Information: Cancer Res 2009;69(23 Suppl):B57.
Abstract B6: Revealing DAPK1 transcriptional regulation by RNA interference screening with an integrated BAC-reporter system
Authors: Oakes Christopher C. , Park Yoon Jung , Boutros Michael , Plass Christoph
Published: May 2012

High-throughput RNA interference screening has been used successfully to reveal novel gene function. Currently used expression reporter systems employ small constructs of synthetic or endogenous sequences containing pathway-specific response elements. The use of these small reporter systems does not allow for the recapitulation of the regulatory complexity that controls an individual gene of interest. Death-associated protein kinase 1 (DAPK1) is a tumor suppressor gene that is silenced in a broad variety of cancers. Specifically, we have previously found DAPK1 to be silenced in virtually all cases of chronic lymphocytic leukemia and silencing is associated with promoter DNA methylation. Although epigenetic silencing is indicative of a transcriptional defect, the basis for this silencing is unknown. Here, we have employed a novel approach to elucidate genes involved in the transcriptional regulation of DAPK1. Using BAC recombineering and stable transgene integration, we have engineered human cells to express a DAPK1-luciferase reporter under the control of approximately 180 Kb of sequence surrounding the DAPK1 transcriptional start site. By performing genome-wide siRNA high-throughout screening, we have been able to determine candidate genes involved in DAPK1 regulation. We have also performed screens in parallel using small, plasmid-based promoter reporter constructs by transient transfection and stable integration. Evaluation of these reporter systems has revealed significant drawbacks of plasmid-based reporters which include nonspecific effects arising from proximal vector backbone sequences. In conclusion, an integrated BAC-reporter system is useful for the assessment of gene-specific transcriptional regulation. Future work will involve determining the disease-specific roles for novel DAPK1 regulatory genes.

Citation Information: Cancer Res 2009;69(23 Suppl):B6.
Abstract B67: A mechanistic link between whole-chromosome aneuploidy and DNA damage
Authors: Crasta Karen , Nezi Luigi , Hennessy Morgan , Cameron Lisa , Pan Yunfeng , Chowdhury Dipanjan , Pellman David
Published: May 2012

Cancer is associated with aneuploidy—both structural defects in chromosomes and abnormal numbers of intact chromosomes. The contribution of chromosome breaks to tumorigenesis is well accepted because of the possibility of generating cancer-causing mutations. However, the role of alterations of intact chromosome numbers remains a subject of debate, principally because mechanisms by which whole chromosome aneuploidy would affect cancer pathogenesis are poorly understood. We now have evidence to support one mechanism by which whole chromosome aneuploidy can lead to DNA breaks, via mis-segregation of chromosomes into micronuclei, structures commonly observed in cancer cells. Micronuclei containing intact chromosomes can form after mitotic errors, when the nuclear envelope reforms around a lagging chromosome. We have developed procedures to monitor the fate of micronuclei. Strikingly, whole chromosome-containing micronuclei develop evidence of DNA breaks during the first S phase after they are formed. Blocking DNA replication blocks the formation of these breaks. Defective DNA replication may be due to defective nucleocytoplasmic transport because micronuclei display a marked defect in assembling nuclear pore complexes. These findings suggest one mechanism by which mitotic errors result in DNA damage and thus potentially promote tumorigenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):B67.
Abstract B69: SUMO-binding proteins as effectors and regulators of SUMO modification
Authors: Guzzo Catherine M. , Uzoma Ijeoma , Zhu Heng , Cox Eric , Blackshaw Seth , Matunis Michael J.
Published: May 2012

SUMOs (Small Ubiquitin-related Modifiers) are reversibly conjugated to other proteins in the cell, thereby regulating a wide range of essential functions. Many of the functions regulated by SUMOylation are intimately tied to processes directly relevant to cancer, including maintenance of genome integrity and cell cycle regulation. Studies in the past several years have in fact linked SUMOylation to a variety of human cancers, including leukemia, prostrate, breast, and colon cancers. In addition, SUMOylation has a direct role in promoting cancer metastasis. Thus, developing a fundamental understanding of the signals and factors that regulate SUMOylation, as well as how SUMOylation affects protein function, has the potential to lead to important new insights into cancer development, diagnosis, and treatment. We hypothesize that the three SUMO paralogues expressed in vertebrate cells (SUMO-1, SUMO-2 and SUMO-3), as well as their polymeric forms, represent unique signals recognized by unique proteins. Moreover, we propose that SUMOylation often promotes interactions between modified proteins and downstream factors containing SUMO-interacting motifs (SIMs). To test this hypothesis, we have initiated high throughput studies to identify novel SUMO-1, SUMO-2 and SUMO-2 polymeric chain binding proteins. Using protein microarray analysis, we identified ∼400 mostly nuclear proteins, many with preferential affinity for specific SUMO paralogues. Among these 400 putative SUMO-binding proteins was a family of zinc finger proteins that we have determined to possess a novel SIM. This family includes human proteins ZNF261, ZNF198, ZNF237, ZNF262, and ZNF258 and each protein contains a stretch of unique tandem zinc fingers called MYM (myeloproliferative and mental retardation) domains. Although the exact functions of MYM-type zinc finger motifs are not known, proteins containing these motifs are linked to myeloproliferative syndromes, X-linked mental retardation, and transcriptional regulation. We have found that although ZNF261 contains four canonical SIMs, these SIMs do not mediate SUMO binding. Rather, analysis of a series of truncation mutants of ZNF261 revealed that the MYM-type zinc finger motifs are both necessary and sufficient for SUMO-binding. MYM-type zinc finger motifs represent only the second known class of SUMO-binding motifs.

Citation Information: Cancer Res 2009;69(23 Suppl):B69.
Abstract B74: A microRNA expression signature of response to EGFR inhibition targets EMT in both lung and pancreatic cell lines
Authors: Balko Justin M. , Coakley Virginia L. , Britson Joel S. , Willian Michael C. , Frolov Andrey , Black Esther P.
Published: May 2012

Purpose: The intent of the study described herein is to develop and characterize a microRNA gene expression signature of response to EGFR inhibition portable to solid tumors for which treatment with an EGFR inhibitor is indicated.

Experimental Design: Treatment with inhibitors of the epidermal growth factor receptor (EGFR) can result in clinical response in both non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC), but only in a minority of unselected patients. Identification of biomarkers that can direct treatment to the responsive population is essential and here we describe generation of a microRNA gene signature of response to EGFR inhibition. Using a Taqman MicroRNA Array of 381 probes, we demonstrate that a 20-gene microRNA signature derived from NSCLC cell lines can predict sensitivity of both lung and pancreatic cancer cell lines to erlotinib, a small molecule inhibitor of EGFR. Overlap of independently generated microRNA expression signatures of NSCLC and pancreatic cell lines produces a fourgene signature. We characterize a target of the reduced signature, ZEB1, in NSCLC and pancreatic cancer cell lines by evaluation of mRNA and protein expression. We evaluated ZEB1 protein and the ZEB1 target, E-cadherin, after transfection of mir-200c into inhibitor-resistant NSCLC cell line, A549.

Results: We have identified a 20-gene microRNA signature of response to EGFR inhibition that predicts both inhibitor-sensitive NSCLC and pancreatic cell lines. By overlapping independent signatures of response in NSCLC and pancreatic cancer cell lines, a four gene microRNA signature is identified that targets proteins responsible for epithelial to mesenchymal transition (EMT). Of those proteins, ZEB1 is a transcription factor responsible for repression of E-cadherin expression. Both ZEB1 mRNA and protein expression are down-regulated in erlotinib-sensitive NSCLC in which the microRNA genes 200b, 200c, and 141 are up-regulated. Ectopic expression of mir-200c in inhibitor-resistant lines induces cell death, suggesting that down-regulation of this microRNA is essential for viability in these NSCLC lines.

Conclusions: The 20-gene miRNA signature of sensitivity provides a means for defining response that can be generated from FFPE samples of tumor rather than fresh tumor samples. Whereas mRNA is difficult to isolate from FFPE samples due to degradation, microRNA may survive the fixation process. Thus, response to EGFR inhibition in the second- or third-line can be predicted from fixed tumor samples collected early in the treatment of an individual patient, eliminating the need for acquisition of fresh tumor. Further, the microRNA signature identifies the well-studied EMT pathway indicative of EGFR-inhibitor sensitivity and provides biological significance for the members of the signature.

Citation Information: Cancer Res 2009;69(23 Suppl):B74.
Abstract B77: Chk1-Lats2-14-3-3 is a novel signaling pathway that regulates the P-body formation in response to DNA damage
Authors: Okada Nobuhiro , Yabuta Norikazu , Nojima Hiroshi
Published: May 2012

Purpose: Lats2 (Large tumor suppressor 2), a member of the conserved Lats kinase family, is a human tumor suppressor gene. We previously reported that Lats2 knockout in mice displays embryonic lethality and the MEFs from Lats2−/− mice causes mitotic defects including centrosome fragmentation and cytokinesis defects associated with nuclear enlargement and multinucleation. DNA damage response (DDR) plays an essential role in maintaining the integrity of the genome and is crucial for cancer avoidance. The purpose of this study is to investigate the role of Lats2 in DDR, which prevents chromosome instability and malignant progression of human cancer by keeping the chromosome stability. MicroRNAs (miRNAs), endogenous RNAs of approximately 22 nucleotides, play an important role in the regulation of gene expression: miRNAs are incorporated into miRNA-induced silencing complexes (miRISCs), which leads to either cleavage or translational repression of targeted mRNAs. mRNA processing bodies (P-bodies) are sites where untranslated mRNAs accumulate, and mRNA degradation, translational repression, and mRNA surveillance take place.

Methods: We raised two phospho-specific antibodies against the phosphorylation sites of Lats2 by Chk1 and 14-3-3 by Lats2 to analyze the phosphorylation status and localization by Western blot and immunocytochemical analyses. Moreover, we established myc-tagged U2OS/siLats2 strains that stably expressed Lats2 mutated the phosphorylation site to alanine or aspartic acid, and examined the effects of those on DNA damage signaling pathway.

Results: We found that Lats2 is phophorylated by Chk1 and/or Chk2 in response to DNA damage, which induced the phosphorylation of 14-3-3 by Lats2. Immunocytochemical analysis using a phospho-specific antibody against 14-3-3 revealed that the phosphorylated 14-3-3 localized to the P-body, and these signals are enhanced in response to DNA damage. We propose here that 14-3-3 is a novel regulatory protein for P-body formation that promotes the miRNA-mediated gene silencing. Indeed, depletion of 14-3-3 or Lats2 reduced the localization to the P-body of both 14-3-3 and a putative P-body scaffold protein, GW182. Eventually, this pathway regulates the translesion synthesis (TLS), a process that synthesizes DNA across DNA lesion, through p21 translational repression by miRNA.

Conclusion: We conclude that Chk1-Lats2-14-3-3 axis is a novel DNA damage signaling pathway that regulates the P-body assembly, thereby causing the translational repression of target genes via miRNA. These results provide the first evidence for a mechanism in which the DNA damage response pathway regulates miRNA-mediated translational repression through P-body assembly.

Citation Information: Cancer Res 2009;69(23 Suppl):B77.
Abstract B8: The two isoforms of ADAM9 have opposing roles in breast cancer cell migration
Authors: Fry Jessica L. , Toker Alex
Published: May 2012

Tumor cell migration is a complex event mediated by the genetic characteristics of the tumor cells themselves in cooperation with changes in the tissue microenvironment. ADAM9, a member of a family of transmembrane matrix-metalloproteases named for their integrin-binding Disintegrin And Metalloprotease domains, is implicated in cell-cell and cell-matrix interactions as well as in shedding of receptors and ligands. ADAM9 is present in breast cancer tumors and cell lines as two alternatively spliced isoforms—ADAM9-L and the truncated, secreted, ADAM9-S. We find that ADAM9-S is expressed primarily in the more migratory basal subtype of breast cancer cell lines, and exogenous and endogenous overexpression of ADAM9-S increases breast cancer cell migration, leading to our hypothesis that ADAM9 present in the breast tumor environment mediates tumor cell migration. Using lentiviral-shRNA constructs, we silenced expression of endogenous human ADAM9 protein in the BT549, ZR75-1 and CAMA-1 breast cancer cell lines, resulting in an increase in cell migration measured by transwell assay. This enhanced migration is reversed by reintroduction of ADAM9-L protein into these cells, while reintroduction of ADAM9-S enhances this migration phenotype in a metalloprotease-dependent manner, showing opposing roles for the two isoforms of ADAM9 in breast cancer cell migration. Furthermore, analysis of functional domain mutants of ADAM9-L shows that ADAM9-L suppresses cell migration in a metalloprotease-independent manner. We also find that ADAM9-L expression alters the migratory phenotype of α-6 integrin-blocking antibody GOH-indicating a role for ADAM9-L in integrin signaling. Future analysis of the proteolytic substrates of ADAM9-S, as well as the subcellular localization and downstream signaling of ADAM9-will further illuminate the mechanisms by which ADAM9 isoforms differentially mediate cell migration.

Citation Information: Cancer Res 2009;69(23 Suppl):B8.
Abstract C11: CD117 and Stro-1 identify osteosarcoma stem cells associated with metastasis and chemotherapy-drug resistance
Authors: Adhikari Amit S. , Agarwal Neeraj , Wood Byron M. , Porretta Constance , Ruiz Bernardo , Pochampally Radhika , Iwakuma Tomoo
Published: May 2012

Osteosarcoma, the most common bone cancer, is the second highest cause of cancer-related death in children and adolescents. Approximately 90% of cases show micro-metastasis at diagnosis, making systematic chemotherapy the first choice of treatment. Despite intensive chemotherapy, the survival rate for high-grade osteosarcomas remains only 50-80%. The inadequacy of current treatments may result from the inability to effectively target cancer stem cells (CSCs) or tumor initiating cells (TI-Cs) of osteosarcoma. Thus, novel therapies targeting cancer stem cells (CSCs) in osteosarcoma is an urgent requirement for eradicating this dreadful disease. Here, we have identified mouse and human osteosarcoma CSCs using mesenchymal stem cell markers CD117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells. CD117+Stro-1+ cells efficiently formed serially transplantable tumors, whereas CD117-Stro-1-cells rarely initiated tumors. Upon orthotopic injections, CD117+Stro-1+-derived tumors metastasized at a high frequency. Further, CD117+Stro-1+ cells were capable of differentiating into adipogenic lineage, and were enriched for CXCR4 and ABCG2, respectively associated with metastasis and drug-resistance. Thus, CD117 and Stro-1 identify osteosarcoma CSCs with the most lethal characteristics of the disease—metastasis and drug-resistance—proposing these markers as potential candidates for CSC-targeted drug delivery towards eradication of osteosarcoma.

Citation Information: Cancer Res 2009;69(23 Suppl):C11.
Abstract C12: EGFR signaling controls the chromatin binding status of BMI1 through a CK2/PP1 phosphorylation/dephosphorylation pathway and affects astrocytoma proliferation and neural stem cell differentiation
Authors: Hawes Jessica H. , Tuskan Robert G. , Agaki Keiko , Reilly Karlyne M.
Published: May 2012

Studies of brain tumors demonstrate the importance of a rare tumor cell population with the capability of regenerating tumors upon transplantation. These cancer stem cells (CSC) are believed to be more resistant to therapy than the bulk of brain tumor cells. Studies in mouse brain tumor models demonstrate that neural stem cells (NSC) can be an important initiating cell type for brain tumors, but the relationship between NSC and CSC is unclear. EGF is a critical growth factor for NSC maintenance in culture and EGFR signaling is upregulated in glioblastoma. Similarly, the chromatin modulator BMI1 is necessary for NSC maintenance, and has been implicated in glioblastoma. We present studies linking cell surface EGF signaling and nuclear BMI1 chromatin binding. We demonstrate that BMI1 is specifically phosphorylated by EGFR signaling using EGF and TGFα, but not by other ligands tested. We have identified three amino acids encompassing the phosphorylation site in response to EGFR signaling, and show that CK2 and PP1 are necessary for control of BMI1 phosphorylation. This phosphorylation alters the chromatin binding state of BMI1 and transcriptional regulation in astrocytoma cells. Interestingly we find that BMI1 can alter transcriptional profiles in both EGF-dependent and independent ways, suggesting that different signaling pathways could have specific effects on changing the BMI1-dependent epigenetic state of subsets of genes. Furthermore, EGF-induced phosphorylation of BMI1 is important for astrocytoma growth in vitro and in vivo, as well as maintenance of NSC.

Our data link signals outside the cell to the nucleus important for NSC maintenance and astrocytoma proliferation. These data represent a common pathway in NSC and CSC that may explain the importance of EGF signaling for NSC maintenance and gliomagenesis, and suggest a novel downstream pathway (CK2/PP1) involved in these processes.

Citation Information: Cancer Res 2009;69(23 Suppl):C12.
Abstract C20: MGMT promoter methylation and analysis of microsatellite instability in head and neck cancer by consensus markers
Authors: Dalay Nejat , Yalniz Zubeyde , Demokan Semra , Suoglu Yusufhan , Ulusan Murat
Published: May 2012

Head and neck cancer is the sixth most common cancer in the world and traditional prognostic indices are of limited use in predicting prognosis. Therefore, there is a need for a beter understanding of the molecular pathogenesis of the disease and a great effort is being made to determine new prognostic and predictive markers that could improve stratification of the patients

Polymorphic microsatellites are useful genetic markers and important tools for studying genetic alterations in tumors. Microsatellite instability refers to the impairment of the mismatch repair system resulting in gains or losses in the number of the tandem repeat units. Cells with mutated mismatch repair genes are not able to correctly repair these errors. The importance of microsatellite instability in head and neck cancer is not well established because of lack of comparable parameters, use of different markers, different criteria in classification and discrepancies concerning the loci examined. Recently it has been shown that mononucleotide markers are more sensitive and specific than dinucleotide markers for the detection of microsatelite instability. No standard approach for the study of MSI has been agreed to in HNC. In this study we analyzed the role and incidence of microsatellite instability in head and neck cancer with criteria and markers recommended by NCI which have been widely used for different types of cancer. We also analyzed methylation of the promoter region of the MGMT gene which functions in the repair pathway of alkylating nitrosocompounds. Microsatellite instability was analyzed in matching tumor and normal tissue samples from 99 patients with head and neck cancer using a panel of five (NR21, BAT25, BAT26, NR24 and MONO27) mononucletoide markers. Two pentanucleotide markers were used to correctly identify the individual tumor and normal tissue samples. The analyses were performed on an ABI Prism 310 capillary electrophoresis system. Microsatellite instability was observed in 26 patients (26.2%). The most frequently affected markers were BAT 25 and NR21 which displayed changes in repeat length in 19 and 11 patients, respectively. Among the microsatellite-positive patients 17 tumor samples (17.1%) displayed high degrees of instability while the remaining 9 samples were instable in only one locus. In five patients instability was observed in three or more loci. Instability was more frequent in tumors with advanced stages. Significant methylation of the MGMT gene was observed in most patients. In 31 patients both alleles were methylated, while in 65 samples partial methylation was observed.

Citation Information: Cancer Res 2009;69(23 Suppl):C20.
Abstract C23: Therapy of triple-negative breast tumors by targeting the MYC oncogene pathway
Authors: Horiuchi Dai , Kusdra Leonard , Klein-McDowell Molly , Creasman Katelyn J. , Goga Andrei
Published: May 2012

Human breast cancers that lack expression of the estrogen and progesterone nuclear receptors and with low HER2 expression are known as “gtriple-negative” tumors. These are amongst the most aggressive breast cancers and no targeted therapeutics exists for this subclass of human tumors.

MYC is a proto-oncogene transcription factor that plays a key role in cell cycle transition of mammalian cells. Increased expression of MYC is estimated to occur in ∼ 20–50% of human breast cancers. Genomic amplification of the MYC locus, 8q24, is correlated with decreased patient survival, and MYC-over-expression in breast cancer cells has been associated with a resistance to anti-hormonal therapies that seek to inhibit ER signaling. Furthermore, MYC has recently been shown to be one of the genes unique to high-grade mammary tumors. Thus, these prior studies suggest that the level of MYC expression might be useful as a biomarker and that the inhibition of MYC function may be a useful therapeutic approach against this difficult-to-treat type of breast cancer. However, while MYC is strongly implicated in breast tumorigenesis, no direct small molecule inhibitors of its transcriptional activity are available in clinical practice.

An alternative approach to directly inhibiting MYC is to find “synthetic-lethal” combinations that selectively kill those cells with elevated MYC levels without affecting normal cells. The term “synthetic-lethality” refers to a genetic context in which a mutation in a gene, which itself does not cause lethality, can cause lethality when combined with a mutation in another gene.

Using engineered cells, we have previously demonstrated that inhibiting cyclin-dependent kinase 1 (CDK1) can kill cells that over-express MYC. We have now used a panel of breast cancer cell lines established from human patients to show that (1) those breast cancers that are receptor-negative have significantly higher levels of MYC protein, (2) that the MYC pathways are critical for the survival and proliferation of this tumor subset, (3) CDK1 inhibition in triple-negative breast cancer cells results in the initiation of apoptosis mediated by mitochondria-dependent intrinsic pathway, and that (4) this apoptotic event is specifically achieved by altering the balance between pro-survival and pro-apoptotic members of the Bcl-2 family proteins. Our results present a novel treatment strategy for “triple-negative” breast cancers by targeting the MYC pathways via CDK inhibitors.

Citation Information: Cancer Res 2009;69(23 Suppl):C23.
Abstract C25: A five-gene signature is associated with tumor progression and poor prognosis in colorectal cancer
Authors: Hao Jummei , Li Guangqiu , Sui Hongmei , Si-Ma Xueqing , Liu Chao , Ding Yanqing , Li Jianming
Published: May 2012

In order to develop an experimentally derived molecular signature closely associated with survival of colorectal cancer (CRC) patients from mouse tumor models, we isolated single cell-derived progenies (SCPs) from SW480 CRC cell line and compared their metastatic potential in an orthotopic implantation model of murine CRC. We compared the differences in the gene expression profiles of a high metastatic variant SCP51, a low metastatic variant SCP58 and their parent SW480/EGFP. We found that 143 genes were commonly differentially expressed between SCP51 and SCP58 cells, SCP58 and SW480/EGFP. Using Gene-annotation enrichment analysis of DAVID, 80 genes were found in top ten clusters of the analysis. Thirty-two genes of above the 80-gene set were found to be involved in metastasis by Genclip, a text mining program. We selected five genes (LYN, SDCBP, MAP4K4, DKK1 and MID1) for immunohistochemical analysis in a cohort of 181 CRC patients. Results showed those LYN, SDCBP, MAP4K4, MID1 and five-gene signatures were significantly correlated with overall survival in patients with CRC. We also found that LYN, MAP4K4 and MID1 but not SDCBP and DKK1 expression was closely correlated with lymph node metastasis and Dukes classification in patients with CRC. Our five-gene signature was closerly associated with survival of CRC patients and could be used as a potential prognostic factor for CRC.

Citation Information: Cancer Res 2009;69(23 Suppl):C25.
Abstract C31: Identification of causal genetic events in oral cancer progression
Authors: Hughes Shevaun E. , Tsui Ivy F. L. , Poh Catherine , Garnis Cathie
Published: May 2012

Background: Oral cancer is one of the world's leading causes of cancer death, with late stage disease diagnoses and high rates of recurrence accounting for poor survival rates. Like many solid tumors, oral cancer develops through a series of histopathological stages from dysplasia, to carcinoma in situ (CIS), to invasive disease. Escalating genomic instability and the accumulation of critical gene alterations is thought to drive disease progression. Causal gene changes remain to be identified for oral cancer. The objective of this study is to determine the essential gene alterations underpinning oral cancer progression.

Hypothesis: Since cancers arise through the accumulation of genetic alterations, gene changes detected in the earliest stages of disease are the foundation of observed cancer phenotypes and essential for oral tumorigenesis.

Experimental Approaches: Oral premalignant lesions and tumor samples were obtained from the Oral Biopsy Service of British Columbia and are associated with well annotated patient information (including disease outcomes). High resolution tiling-path array comparative genomic hybridization was used to define segmental DNA changes for each sample. Recurring regions of DNA alteration detected in premalignant lesions known to progress and preserved in later stage disease were flagged as critical events. Our list of candidate genes within these regions will be refined by analysis of publicly available genomic and gene expression data from oral tumors.

Results: Whole genome copy number profiles from over 100 oral lesions and tumors have been successfully generated using array comparative genomic hybridization. From this data we have identified several molecular subgroups associated with initiation and progression of oral cancer.

Significance: With knowledge of the key genes fueling oral tumorigenesis, we will be able to derive novel biomarkers capable of predicting disease behavior as well as gain a better understanding of the molecular pathways and gene networks responsible for driving oral cancer progression.

Citation Information: Cancer Res 2009;69(23 Suppl):C31.
Abstract C34: Amplified loci on chromosomes 8 and 17 predict Tamoxifen resistance in ER+ breast tumors
Authors: Bilal Erhan , Ganesan Shridar , Bhanot Gyan
Published: May 2012

Most ER+ breast cancers are treated with anti-estrogens like Tamoxifen which disrupt the Estrogen growth pathway. However, not all ER+ cases respond to this therapy. This study aims to identify genetic markers associated with increased sensitivity or resistance to Tamoxifen using outlier analysis. We collected 268 ER+ samples of gene expression data from three separate published studies on Tamoxifen treated patients with follow-up information on distant metastasis-free survival time. Genes were associated with Kaplan-Meyer survival curves based on their outlier profile and filtered, using the long-rank test, for differential survival distributions at p-value<0.05. Additional correlation analysis and PCA clustering were used to identify pathways and chromosomal regions enriched with gene markers correlated with Tamoxifen response. We find significantly over-expressed cell cycle genes correlated with poor survival together with an enrichment of chromosomal regions 17q21.33–q25.1, 17q12, 8p11.2 and 8q24.3. These amplicons were previously documented in breast cancer and contain known oncogenes ERBB2, LSM1, WHSC1L1 and HSF1 associated with more aggressive forms of the disease. Since the majority of samples enriched in the cell cycle pathway also have at least one of the amplicons, this suggests the amplifications on chromosomes 8 and 17 are associated with increased proliferation. In addition to this, a relative Oncotype DX™ score was calculated for samples using normalized expression levels and published weights. This analysis showed that high Oncotype DX™ scores are generated by tumors having either the HER2 amplicon (highest Oncotype DX™ scores), or one of the other amplicons on chromosomes 17 and 8. In contrast, low Oncotype DX™ scores were found only for tumors that do not exhibit any of the identified chromosomal aberrations. This implies that amplicons 17q21.33–q25.1, 8p11.2 and 8q24.3 may be responsible for higher proliferation and poor outcome in the setting of Tamoxifen treatment clinically observed in a subset of ER+, HER2− breast cancers. These results have significant implications for the development of targeted treatments for Tamoxifen-resistent ER+ breast cancers as well as in improving current methods of drug response prediction.

Citation Information: Cancer Res 2009;69(23 Suppl):C34.
Abstract C4: Sp2: A regulator of stem cell differentiation and tumorigenesis
Authors: Kim Tae-Hyung , Horowitz Jonathan M.
Published: May 2012

Transcription factors regulate gene expression and de-regulation of their activities can transform normal cells into tumor cells. The Sp-family of transcription factors consists of nine distinct proteins (Sp1-9) that are evolutionarily-conserved and share a common DNA-binding domain. Sp2 is one of several poorly-characterized members of this transcription factor family. Previous data from our lab and others has indicated that Sp2 is over-expressed in human prostate cancer and in murine skin squamous cell carcinoma. To determine if the de-regulated expression of Sp2 is oncogenic, transgenic mice over-expressing mouse Sp2 via the keratin 5 (K5) promoter were generated. This promoter drives expression of linked genes in basal cells of stratified epithelia, such as skin. Two different transgenic lines (Family A and C) were derived and characterized. The transgenes carried by Family A and C mice were localized to chromosomes 6 and 5, respectively. Endogenous genes were not disrupted by transgene integration in Family A, whereas transgene integration in Family C mice occurred within the latrophilin 3 (Lphn3/CIRL3) locus. Homozygous Family A and C animals develop normally and are indistinguishable from wild-type and hemizygous littermates until post-natal day 2. Family C animals invariably perish at this time due to skin deterioration whereas homozygous Family A animals develop skin lesions on post-natal day 4, are runted and developmentally retarded and perish prior to post-natal day 13. Immunohistochemical staining revealed that that an expanded population of K5-negative, K14-positive progenitor cells inundates all epidermal cell layers of homozygous Family A animals. Expression of markers of differentiated basal keratinocytes (e.g., keratin 10, loricrin) is decreased in homozygous Family A animals. The expression level of keratin 6, a marker of epidermal wounding, inflammation, and/or tumorigenesis is increased in Family A animals beginning at post-natal day 4 as is the expression and distribution of CD34, a hair follicle stem cell marker. Hemizygous Family A animals progress to adulthood unremarkably, however 70% exhibit varying degrees of alopecia (hair loss) beginning at 1 month of age. Family C hemizygotes do not develop alopecia, however an expanded pool of progenitor cells renders these animals susceptible to wound-induced neoplasia, and the incidence of papilloma formation rises with an age-dependent increase in Sp2 levels. Sp2 expression in Family A hemizygotes is insufficient to promote wound-induced neoplastic growth, but epidermal differentiation is slowed sufficiently to retard rates of wound healing. These and other results have led us to speculate that Sp2 functions to limit stem cell differentiation and thus its over-expression contributes to the outgrowth of poorly-differentiated, tumorigenic cells.

Citation Information: Cancer Res 2009;69(23 Suppl):C4.
Abstract C40: Salicylic acid-based small molecule inhibitors of Stat3 show potent and selective activity in a variety of human cancer cell lines
Authors: Page Brent D. G. , Fletcher Steven , Schimmer Aaron , Turkson James , Gunning Patrick T.
Published: May 2012

Signal transducer and activator of transcription 3 (Stat3) protein is an oncogenic transcription factor that exhibits aberrant functioning in many types human cancer. Constitutive Stat3 activation causes over-expression of anti-apoptotic proteins and makes cancer cells resistant to natural apoptotic processes. Inhibiting Stat3 reduces the expression of anti-apoptotic proteins and can selectively kill cancerous cells. We have identified several salicylic acid based inhibitors of Stat3 that show potent activity in vitro and in whole cell assays. In silico docking studies have revealed S3I-201, a known Stat3 inhibitor, exhibits incomplete occupation of Stat3's SH2 domain. A thorough structure activity relationship study has provided a series of compounds that more completely occupy Stat3's SH2 domain and show potent and highly selective activity against Stat3 and a variety of human cancers. Inhibitor design, synthesis, in vitro and in vivo assays and preliminary metabolic studies will be discussed.

Citation Information: Cancer Res 2009;69(23 Suppl):C40.
Abstract C50: Novel silicon derivatives of indomethacin: Generation of potent COX-2 selective agents with improved anticancer activity
Authors: Zakai Uzma I. , Gately Stephen , West Robert
Published: May 2012

Elevated tumor cyclooxygenase-2 is associated with poor overall survival in patients with a variety of cancers. Patients with elevated COX-2 that receive a COX-2 inhibitor have demonstrated improved survival. Indomethacin (IM) is a COX-non-selective inhibitor with demonstrated anticancer activity in patients. The anticancer effects of IM are related to inhibition of COX-2 as well as COX-independent targets. We now report the synthesis and activity of novel IM-sila- amide derivatives where strategic silicon addition results in COX-2 selective IM derivatives that demonstrate activity in mouse models of human cancers and are devoid of the COX-1 associated toxicities. The sila-IM compounds demonstrated improved inhibition of purified human COX-2, IC50 0.2 µM and limited inhibition of COX-1 up to 10 µM. Sila-IM derivatives were also found to be potent inhibitors of NFκB transcription activating function at concentrations below 1.0 µM. A sila-IM derivative Silexsyn was tested in various animal models of human cancer following oral administration. Significant activity was observed in models of non-small cell lung cancer and pancreas cancer. Potent synergism was observed for Silexsyn in combination with EGFR inhibitors. A synthetic methodology incorporating a heteroatom in the amino-functional silane has been developed and used to generate second-generation sila-IM derivatives that could have improved pharmacological properties. The introduction of silicon into an approved scaffold represents an efficient strategy for the rapid production of biologically relevant therapeutics for the treatment of cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):C50.