bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4942: The role of one-carbon metabolism in global and promoter methylation of breast cancer in a population-based study
Authors: Xu Xinran , Gammon Marilie D. , Zhang Yujing , Cho Yoo H. , Wetmur James G. , Bradshaw Patrick T. , Teitelbaum Susan L. , Neugut Alfred I. , Santella Regina M. , Chen Jia
Published: February 2011

Promoter-CpG island hypermethylation, accompanied by global DNA hypomethylation, are common molecular defects in cancer cells. One-carbon metabolism is involved in the DNA methylation process as it provides the universal methyl donor for methylation reactions. Thus, perturbation of one-carbon metabolism might influence DNA methylation status. Using the resource of the Long Island Breast Cancer Study Project, a population-based case-control study, we investigate the relationships of dietary and genetic factors involved in the one-carbon metabolism pathway with LINE-1 methylation level in blood cells as well as promoter methylation status of a panel of breast cancer genes in tumor tissue. LINE-1 methylation level was assessed by Pyrosequencing. Gene promoter methylation was examined by MS-PCR and MethyLight assays. Global methylation measured as LINE-1 methylation level from blood DNA was not associated with risk of developing breast cancer. Neither dietary intakes of methyl-related micronutrients nor genetic polymorphisms in one-carbon metabolism was associated with LINE-1 methylation level. LINE-1 methylation level was higher among cases with methylated APC (p=0.04). Promoter methylation of APC and p16 were correlated (p=0.0007). Higher betaine intake was associated with methylated APC promoter (p, trend=0.01). Cases with the 2R/2R genotype of TYMS are more likely to have a methylated ER promoter (p, trend=0.03). Our results indicate that one-carbon metabolism has a limited influence on DNA methylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4942.
Abstract 4946: The nicotinic acetylcholine receptor genes CHRNA3 and CHRNB4 are epigenetically deregulated in lung tumorigenesis
Authors: Scherf David B. , Sarkisyan Narek F. , Jacobsson Hanna T. , Claus Rainer , Bermejo Justo L. , Muley Thomas , Meister Michael , Dienemann Hendrik , Plass Christoph , Risch Angela
Published: February 2011

Background: Lung cancer, the leading cause of cancer-related death worldwide, is strongly connected with tobacco smoking. Genome-wide association studies have recently identified three lung cancer susceptibility regions in 15q25, 5p15.33 and 6p21.33 that strongly associate with lung cancer risk. Here we investigate whether these three susceptibility regions harbor genes with different DNA methylation patterns in tumor and adjacent normal lung tissue from the same individual.

Methods: For DNA methylation analysis we used the MassARRAY (Sequenom), a MALDI-TOF based method, to quantify DNA methylation in tumor and normal lung tissue from 37 patients. We analysed 17 CpG islands situated in nine different genes situated in the three lung cancer susceptibility regions. Nonparametric and hurdle models were used to evaluate methylation differences. Additionally, reverse transcription PCR was performed with a LightCycler and expression analysis was conducted in 50 tumor/normal pairs. This included 25 adenocarcinoma (AdC) and 25 squamous cell carcinoma (SCC) (10xStage I, 10xStageII, 5xStageIII) matched pairs. In reexpression experiments we treated A549 and H1299 cells with 0.5 and 1 µM of 5-aza-deoxycitidine (5-Aza-dC).

Results: Comparing tumor and normal tissue we obtained significant differences in DNA methylation for at least two CpG units situated in a putative promoter (p<0.001) for each of three genes, the alpha 3 and beta 4 subunit genes of the nicotinic acetylcholine receptor CHRNA3 and CHRNB4 and for the aminoglycoside phosphotransferase domain gene AGPHD. To assess RNA expression of these genes we analyzed 50 tumor/normal pairs by real-time quantitative RT-PCR. Matched pairs analysis showed a significant overall down regulation of CHRNA3 in SCC patient tumors (p=0.002). For CHRNB4 we detected increased transcription in lung tumors in both histologies (p<0.001). The expression analysis for AGPHD did not reveal a clear aberrant deregulation. After drug treatment increased expression was detected for CHRNA3 and CHRNB4 but not for AGPHD transcripts.

Conclusion: In the present study we identified two genes situated in the nicotinic acetylcholine receptor cluster on chromosome 15q25, CHRNA3 and CHRNB4, as being differentially methylated in lung tumors compared to adjacent normal tissue, which is also reflected in gene expression. These results suggest CHRNA3 and CHRNB4 as strong candidate lung cancer susceptibility genes for further investigation in functional analyses to bring gene deregulation into a chronological context in lung tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4946.
Abstract 4947: Homeobox gene HOPX is epigenetically silenced cancer-specifically in colorectal cancer and predicts poor prognosis in stage III colorectal cancer
Authors: Katoh Hiroshi , Yamashita Keishi , Ooki Akira , Sidransky David , Watanabe Masahiko
Published: February 2011

(Background) Colorectal cancer (CRC) is the second most prevalent cancer, and chemotherapy has dramatically improved prognostic outcome of CRC patients over the past several decades. Nonetheless, CRC remains the fourth leading cause of cancer death world-wide, and stage III patients have a high potential for recurrence even after curative surgery. Homeodomain only protein X (HOPX) is a recently identified homeobox gene whose loss of expression has been reported for several types of cancer by promoter DNA CpG islands methylation, but there is no information in CRC.

(Materials and Methods) HOPX is encoding 3 spliced transcript variants, among which the promoter of the isoform b alone (promoter B) harbors CpG islands. We evaluated the impact of its methylation status of promoter B of HOPX gene in CRC. We evaluated 9 CRC cell lines and primary tumor tissue of 245 CRC patients. The methylation status was evaluated by bisulfate sequencing and quantitative methylation specific TaqMan real-time PCR (quantitative-MSP).

(Result) HOPX b mRNA was silenced or remarkably reduced in expression in 8 of 9 CRC cell lines tested. And the expression was robustly restored by treatment with demethylating agents such as 5-aza-2′-deoxycitidine and/or trichostatin A. On quantitative-MSP, HOPX promoter B hypermethylation was found in 56.6% (56/99) of primary tumor tissues and 0% (0/98) of corresponding normal tissues, if cut-off value was set to 2.89) (specificity reached 100.0%). Such cancer-specific methylation was not associated with prognostic factors representing TNM stage factors in the 100 initial leaning sets. However, patients with HOPX promoter B hypermethylation showed significantly worse prognosis than hypomethylated patients (P=0.03), only in 170 stage III patients which have a high potential for recurrence even after curative surgery. And this tendency was also observed even in dissociative analysis of UICC N factor (P=0.01 in N1, P=0.08 in N2). (Conclusion) Our study indicates that transcriptional silencing of HOPX predominantly results from HOPX promoter B methylation, which is a highly specific marker for CRC and also have a potential as a biomarker for tumor malignancy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4947.
Abstract 4953: Identification of direct target engagement biomarkers for kinase drug discovery using quantitative mass spectrometry: PDK1 case study
Authors: Andersen Jannik N. , Paweletz Cloud P. , Pollock Roy , Nagashima Kumiko , Northrup Alan , Ronald Hendrickson C. , Chi An
Published: February 2011

The human genome encodes some 500 protein kinases and an estimated twenty percent of drugs currently in development target this enzyme class. Because kinase inhibitors frequently bind to the conserved ATP-binding pocket, in vitro kinase selectivity profiling of drug candidates are an integral part of the small-molecule drug discovery process. Likewise, the co-development of pharmacodynamic (PD) biomarkers is critical to drug development as they link drug-target inhibition in cells with biological activity. As such PD biomarkers are essential for the interpretation of in vivo PK/PD/efficacy studies, which ultimately inform medicinal chemistry. Here, we present a universal mass-spectrometry-based approached to discover kinase drug-target engagement biomarkers using the phosphoinositide dependent protein kinase 1 (PDK1) as an example. Western blot analysis using a commercial available phosphosite-specific PDK1 antibody (Ser241) revealed that classical, ATP-competitive PDK1 inhibitors do not dynamically impact PDK1 T-loop phosphorylation in cells, despite cell biochemical inhibition of phospho-AKT(Thr308), a bona fide PDK1 substrate phosphorylation site. However, because receptor tyrosine kinases (RTKs), lipid kinases and other pathways often signal via AKT, the monitoring of phospho-AKT as a PD biomarker for PDK1 is not sufficient for delineating the specificity of small-molecule inhibitors and ascertain the degree of PDK1 target engagement in cells. Here, we used stable isotope labeling by amino acids (SILAC) in cell culture combined with immunoprecipitation of PDK1 protein from cells for quantitative mass-spectrometry-based identification of phosphorylation sites modulated by PDK1 inhibitor treatment. We mapped 10 Serine/Threonine phosphorylation sites on PDK1 including several novel phosphorylation sites. Of these, two PDK1 phosphorylation sites (Ser410 and Thr513) were modulated by pharmacological PDK1 inhibition and the degree of dephosphorylation correlated with inhibitor potency in PDK1 enzymatic assays. By contrast, pharmacological inhibition of PDK1 did not quantitatively modulate PDK1 T-loop phosphorylation (Ser241) consistent with the initial western blot analysis. Altogether, our study provides proof-of-concept for identifying novel kinase target engagement PD biomarkers for PDK1, complementing pathway biomarkers such p-AKT and p-S6RP, which are modulated broadly by PI3K-pathway targeting agents including inhibitors of PI3K/mTOR and RTKs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4953.
Abstract 4954: Development and validation of 50 serum cancer multiplexed biomarkers
Authors: Stephen Laurie L. , Ballard Karri L. , McDade Ralph L. , Mapes James P.
Published: February 2011

Biomarkers are indicators of normal biological processes and can be monitored to look at physiological changes. In cancer, a pattern of biomarkers may be useful for the detection, reoccurrence and response to treatment. We have identified 50 biomarkers implicated in cancer and have developed bead-based multiplexed immunoassays for the Luminex system, defined as the OncologyMAP™. Multiplexing is a cost effective method which allows for the measurement of multiple proteins with little sample volume. The OncologyMAP™ consists of markers of cell damage (Annexin A1, HMGB1), soluble receptors (uPAR, TIE-2), enzymes (PSAT-1, aldose reductase), signaling molecules (calcvasculin, EpCAM)) and growth factors (endostatin, BAFF). All markers showed positive signals in either normal serum and plasma samples or cancer serum. Multiplexed assays were run on a TECAN EVO and showed good precision, linearity and spike recovery. The OncologyMAP™ is a valuable tool for cancer biomarker discovery

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4954.
Abstract 4956: Proteomics-based characterization of pathways involved in tamoxifen resistance in breast cancer cells
Authors: Sanchez Betzabe C. , Johansson Henrik , Lehtiö Janne , Stål Olle , Linderholm Barbro K.
Published: February 2011

Background: Although the introduction of tamoxifen (tam) has contributed to the improved survival of breast cancer patients there are those that relapse despite treatment due to de novo or acquired resistance to the drug. Since the mechanisms involved in tam resistance likely involve various pathways, techniques like mass spectrometry allowing simultaneous analyses of multiple proteins are of great value. Our aim is to identify pathways involved in tam resistance by using a mass spectrometry.

Material and methods: Mass spectrometry based proteomics was used to investigate the proteomic changes associated with tam exposure using a tam-responsive and resistant cell line, MCF7 and MCF7/LCC2, respectively. Cytosolic and nuclear fractions were subjected to a shotgun proteomics approach. This included immobilised pH gradient-isoelectric focusing (IPG-IEF) of trypsinized peptides followed by Maldi-TOF/TOF mass spectrometry. Advanced biostatistics was applied to narrow identified targets to those which were significantly deregulated.

Results: Preliminary results indicate a clear distinction between the nuclear and cytosolic fractions in regards to proteins. Various proteins were differentially deregulated in MCF7 compared to the resistant cell line both at the basal level and following tam treatment. At basal level, glucose-6-phosphate dehydrogenase, fatty acid synthase and keratin 18 were significantly upregulated in resistant cells as compared to parental MCF7 cells. Interestingly, tam treatment in MCF7 cells induced a significant increase in expression of several proteins involved in the p53 pathway and related to Huntingtin (HTT) while downregulating other proteins related to Retinoic Acid Receptor alpha (RARA). In contrast, resistant cells showed upregulation of proteins involved with RARA without exhibiting any specific deregulated proteins related to HTT or p53.

Conclusions: Basal differences in protein expression were observed between the tam-sensitive and resistant cell line. Our results indicate that there are potentially deregulated proteins in the tam-sensitive cell line involved in p53, RARA, and HTT pathways in a manner that differs from the resistant one. The expression of the identified proteins will be further characterized. Understanding of the mechanisms involved in tam resistance can potentially aid in the development of new drugs and serve as prognostic biomarkers.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4956.
Abstract 4958: Analysis of the AKT signaling pathway in prostate cancer using a streamlined Western blot workflow
Authors: Feather-Henigan Kelli , Berry Rachael , Kilmer Greg , Webb Brian
Published: February 2011

Relative protein levels in the Akt pathway were analyzed in metastasized and normal prostate cancer cell lines using a new streamlined Western blot workflow and compared to a traditional Western blot method. Protein expression levels in the Akt pathway are commonly studied using Western blotting; however, current Western blot techniques involve multiple time-consuming steps taking more than 5 hours or even days. We used a streamlined Western blot workflow that combines a rapid 10-minute protein transfer and a one-hour immunoblotting protocol. When using an overnight primary antibody incubation, the protocol requires less than 30 minutes. Importantly, this method does not require the use of additional equipment or consumables and does not limit throughput. These data illustrate that current Western blot techniques involving multiple time-consuming steps can be streamlined to yield comparable results for phospho-specific and total protein targets. Results obtained in the analysis of the Akt pathway yielded comparable sensitivity to traditional Western blotting methods for both low- and high-abundance target proteins.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4958.
Abstract 4963: ERG expression levels reflect functional status of androgen receptor in prostate cancer
Authors: Dobi Albert , Nydam Timothy , Furusato Bungo , Chen Yongmei , Sesterhenn Isabell , McLeod David G. , Petrovics Gyorgy , Srivastava Shiv
Published: February 2011

Background: Expression of the ERG proto-oncogene, is activated in 50-70% of prostate tumors by androgen receptor (AR) mediated signals due to the fusion of AR regulated promoters (primarily TMPRSS2 and to a lesser extent SLC45A3 and NDRG1) to the ERG protein coding sequence. Our previous studies of quantitative expression levels of ERG or TMPRSS2-ERG fusion transcripts have noted that relatively low or no ERG expression in prostate tumors significantly associated with progressive disease. Here, we have tested the hypothesis that ERG expression levels in prostate tumor cells reflect AR transcriptional regulatory function in a given biological context of the tumor progression. Therefore, tumors with lower ERG may represent a subset with attenuated AR signaling.

Experimental procedures: Expression of ERG and other AR regulated genes were evaluated in a GeneChip dataset obtained from a panel of laser capture micro-dissected well/moderately differentiated (WD) or poorly differentiated (PD) tumor cells derived from primary tumors of patients, who had no prior androgen ablation treatment.

Results: ERG expression pattern was similar to that of other AR regulated genes. Low frequency of ERG expression was noted in PD tumor cells (30%) in comparison to WD tumor cells (80%), suggesting for subdued AR function in a significant fraction of tumors with genomic alterations of ERG.

Conclusions: We developed a cumulative AR Function Index (ARFI) by integrating ERG into a panel of defined AR target genes, may have future potential in stratifying patients for targeted therapy on the basis of overall AR functional status in primary tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4963.
Abstract 4966: BORIS binding to the promoters of cancer testes antigens, MageA2 and MageA3, causes transcriptional activation of these genes in lung cancer
Authors: Bhan Sheetal , Negi Sandeep S. , Glazer Chad , Chuang Alice , Sun Wenyue , Gaykalova Daria , Shao Chunbo , Ha Patrick K. , Califano Joseph A.
Published: February 2011

BORIS or CTCFL (Brother of the regulator of imprinted sites) is an 11 zinc-finger transcription factor that is involved in epigenetic gene regulation. Boris is normally expressed only in the testes but is aberrantly expressed in many cancers. In the present study, we wanted to study the role of BORIS in the regulation of the cancer testes antigens (CTA) MageA2 and MageA3 in lung cancer. CTAs are also normally expressed in the testes but are upregulated in a variety of cancers like lung, bladder and liver cancers and melanomas. We first investigated the in vivo binding of BORIS to the promoter regions of MageA2 and MageA3 by chromatin immuno-precipitation (ChIP). We found that BORIS was enriched at the promoter regions of these genes in H1299, a lung cancer cell line, compared to the normal lung cell line, NHBE (normal human bronchial epithelial). Interestingly, we found that these genes have high expression in H1299 cell line which is also a high expressor of BORIS. On the other hand, they have very low or no expression in NHBE cells which are also very low BORIS expressors. When BORIS was knocked down in H1299 cells, the expression of these genes was reduced by 25-30%. We then used Luciferase reporter assay to see if MageA2 and MageA3 promoters were activated by BORIS. When BORIS was expressed in the non-expressing cell line NHBE, the activity of the promoters was increased as determined by the luciferase activity. Upon knock-down of BORIS in H1299 cells, the activity of the MageA2 and MageA3 promoters reduced significantly. We further investigated if BORIS binding changes the pattern of histone modification at these promoters. We found that BORIS binding at these promoters in H1299 cells correlates with the enrichment of acetylated H3K9, a modification that is involved in opening up chromatin regions. In the absence of BORIS binding in NHBE cells, methylated H3K9, which is involved with more closed chromatin conformation, is enriched at these sites. These data suggest that BORIS is a positive regulator of the cancer testes antigens and may be a key effector involved in their derepression in lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4966.
Abstract 4967: Changes in c-Myb specificity and activities during the cell cycle in T-ALL and AML
Authors: Quintana Anita M. , Pena Janeth J. , Ness Scott A.
Published: February 2011

The c-Myb transcription factor is a critical regulator of proliferation and stem cell differentiation, and mutated alleles of c-Myb are oncogenic, but little is known about the activities of c-Myb during the cell cycle. Previous studies showed that c-Myb interacts with the cell cycle regulators Cyclin D1 and Cyclin Dependent Kinases 4 and 6, suggesting that c-Myb activity is regulated during the cell cycle. We set out to test that directly, using Chromatin ImmunoPrecipitation (ChIP) assays capable of assessing the activities of c-Myb in primary and flow sorted populations of cells. We performed ChIP assays with cells sorted into each phase of the cell cycle to determine c-Myb specificity and correlated c-Myb occupancy with the relative expression of a number of known target genes. Briefly, asynchronously growing cells were fixed, stained with Hoechst 33342, then flow-sorted into different cell cycle phase populations before processing the samples for ChIP assays. The c-Myb protein associated with different cyclin gene promoters in different stages of the cell cycle, showing that c-Myb specificity and activity are dynamic. By using several different anti-Myb antibodies, we confirmed that the changes were due to relocalization of c-Myb, and not due to changes in the accessibility of individual epitopes. We also analyzed the Myb-regulated CXCR4 gene promoter in both primary AML and Jurkat T-ALL cells. c-Myb occupied the CXCR4 gene promoter during S and G2/M phase of the cell cycle, which correlated with increased CXCR4 expression in cycling cells. In conclusion, our data demonstrate that c-Myb specificity is dynamic, that c-Myb associates with different genes in different phases of the cell cycle and that c-Myb is cell cycle regulated in both T-ALL and primary human AML.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4967.
Abstract 4969: Neuropilin-2 is downregulated by Snail during EMT in lung cancer
Authors: Roche Joëlle , Nasarre Patrick , Potiron Vincent , Nair-Menon Joyce , Gemmill Robert , Drabkin Harry
Published: February 2011

Neuropilins (NRP1 and NRP2) are receptors for class-3 semaphorins that are guidance molecules involved in nerve growth patterning, tissue development, immune response, angiogenesis, and cancer development. Upon semaphorin binding, neuropilins transmit signals to the co-receptor, the plexins. Interestingly, neuropilins are also receptors for VEGF. Semaphorins control cell protrusion, spreading and adhesion and can trigger migratory responses often in a repulsive manner but also attractive depending on the cellular context.

We previously cloned SEMA3F, coding for a class-3 semaphorin, in the chromosome 3p21.3 region which undergoes frequent loss of heterozygosity (LOH) in lung cancer. We further reported that levels of SEMA3F inversely correlated with tumor aggressiveness in human lung cancer and we demonstrated the tumor suppressor activity of SEMA3F in experimental xenograft models. In addition, SEMA3F is directly downregulated by ZEB1, a transcription factor involved in the epithelial to mesenchymal transition (EMT).

Recently, we found an inverse correlation between the transcription factor Snail and NRP2 expression in a panel of 19 lung cancer cell lines. Interestingly, Snail is induced during EMT and NRP2 is the best affinity receptor for SEMA3F. Data will be presented with the H358 NSCLC cell line model where Snail induction decreases NRP2 expression both at the RNA and protein levels. Chromatin immunoprecipation (ChIP) experiment suggests Snail direct binding to the NRP2 promoter. In addition, in lung cancer tissue microarrays, Snail and NRP2 stainings are frequently mutually exclusive.

Our results suggest that loss of SEMA3F and NRP2 would trigger EMT and promote metastasis. Therefore, targeting the semaphorin/neuropilin pathway would be an interesting therapeutic approach in lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4969.
Abstract 497: Mitochondria-targeting photosensitizers activable by two-photon absorption
Authors: Ngen Ethel J. , Xiao Lixin , Xingzhong Yan , You Youngjae
Published: February 2011

Two-photon absorption (TPA) is a non-linear optical process, used to activate photosensitizers in the NIR region (700 - 850 nm) thus affording greater tissue penetration in photodynamic therapy (PDT). Since the efficiency of this process has a quadratic dependence on the light intensity, the photodynamic effects are restricted to a small area around the laser's focal point, thus providing spatial selectivity. Although most clinically approved photosensitizers can be activated by TPA, they generally have very small cross-sections (generally below 10 GM around 800 nm). Thus, light intensities required to activate them could damage normal surrounding tissues. The TPA of photosensitizers can however be improved, without perturbing their ability to generate singlet oxygen, by conjugating them to effective TPA chromophores. These then serve as TPA antennae and upon activation transfer their energy to the photosensitizer which then generates singlet oxygen for PDT. In this study a porphyrin-rhodamine B dyad (TPP-Rh), previously demonstrated to target mitochondria, was evaluated as a two-photon photosensitizer for PDT. TPP-Rh, was synthesized by conjugating a monohydroxy porphyrin photosensitizer (TPP-OH) to rhodamine B (Rh B), an effective TPA chromophore, via a saturated hydrocarbon linker, thus enhancing intramolecular FRET between the dyad components and ultimately the photosensitizer's TPA. To evaluate the efficiency of TPP-Rh as a two-photon photosensitizer we studied both its one and two-photon absorption photophysical properties in comparison to those of its individual components: Rh B and TPP-OH. These included: FRET kinetics, FRET dynamics, and the singlet oxygen generation kinetics. A FRET rate of 1.679 × 1012 s−1 and a 99 % FRET efficiency was calculated for TPP-Rh from its spectral properties suggesting efficient FRET from the TPA moiety (Rh B) to the photosensitizer moiety (TPP). In addition to this, TPP-Rh produced singlet oxygen at comparable rates when excited with both TPA at 800 nm and one photon absorption (OPA) at 650 nm. Thus, this indicates that conjugating photosensitizers to rhodamine B could provide several advantages in PDT: deeper tissue penetration, spatial selectivity and selective photosensitizer accumulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 497.
Abstract 4971: WBP2: A novel phospho-oncoprotein in estrogen receptor-positive breast cancer
Authors: Lim Shen Kiat , Prioux Magali Orhant , Tan Kah Yap , Lim Yoon Pin
Published: February 2011

WBP2 (WW-domain Binding Protein 2) was first implicated in breast cancer through our previous phosphoproteomics screen on the MCF10AT xenograft-derived isogenic cell line model of human breast cancer progression, where WBP2 was found to be differentially phosphorylated during breast cancer development. We had also shown it to be an authentic tyrosine phosphorylation target of EGFR signaling. In a follow up study, we found that WBP2 expression was low or undetectable in normal breast epithelial cells but overexpressed in breast cancer progression model as well as multiple breast cancer cells of various subtypes. Therefore, we aimed to investigate the oncogenic role of WBP2 in the growth and progression of human breast cancer through its protein overexpression and tyrosine phosphorylation. We first mapped the EGF-dependent tyrosine phosphorylation sites on WBP2 and identified a member of the Src family of tyrosine kinases as the putative upstream kinase of WBP2. Next, we found that Estrogen (E2) stimulation could similarly induce tyrosine phosphorylation of WBP2 via a crosstalk between EGFR and ER pathways. Subcellular fractionation study revealed a phosphorylation-dependent nuclear entry of WBP2 upon E2 or EGF stimulation. Consistent with the reported role of WBP2 as a coactivator for nuclear hormone receptor (ERα/PR), abrogation of tyrosine phosphorylation on WBP2 significantly impaired the E2-induced ER transactivation through reduced E2/EGF-stimulated nuclear entry of WBP2 and in vivo E2-dependent WBP2-ER interaction. Stable overexpression of wild type and phospho-mimic mutant of WBP2 in ER-positive MCF7 breast cancer cell enhanced its cell proliferation, anchorage-independent growth in soft agar, cell migration and cell invasion in the E2-independent and/or E2-dependent manner. These cellular processes could be inhibited, at least in part, by the phosphorylation-defective mutant of WBP2. Potential mechanisms of WBP2-mediated oncogenic transformation will be presented. Collectively, our findings have uncovered the potential of WBP2 as a novel phospho-oncoprotein in ER+ breast cancer. It is conceivable that WBP2 and/or its tyrosine phosphorylated form could serve as a potential prognostic marker or therapeutic target for human breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4971.
Abstract 4972: Putative mechanism of TAP63α regulation of chondrocyte maturation during long bone development
Authors: Li Feifei , Lu Yaojuan , Abbassi Sam , Ding Ming , Wu Guojun , Wang Siying , Zheng Qiping
Published: February 2011

p63 is the mouse homologue of the well-known tumor suppressor P53. While it remains controversial as to whether P63 functions as a tumor suppressor gene or an oncogene, mouse genetic studies and mutation analysis of human SHFM syndrome (Split Hand/Foot Malformation) have clearly demonstrated that P63 plays an important role in skin and limb development. The truncated limbs observed in p63 null mice suggest that P63 may contribute to long bone development by playing a role during endochondral bone formation. However, how P63 regulates target gene expression and is linked to endochondral ossification remains largely unknown. Here we present some preliminary data suggesting that P63 may regulate type X collagen gene (Col10a1) expression, thereby impacting the process of chondrocyte maturation.

The type X collagen gene (Col10a1) is specifically expressed in hypertrophic chondrocytes, a critical cell stage linking both skeletal development and multiple skeletal diseases. We have previously shown that a 150-bp Col10a1 cis-enhancer is sufficient to mediate its cell-specific expression in vivo. Our further in vitro studies suggest that Runx2, a master transcription factor for osteoblast differentiation as well as for chondrocyte maturation, is one of the major factors that regulate Col10a1 expression via interaction with its tandem repeat Runx2 binding sites. Interestingly, by yeast one-hybrid approach using this cis-enhancer as bait, we identified p53 related proteins as candidate factors that may contribute to the regulation of Col10a1 expression. We have performed Real-time RT-PCR to examine p63 and Runx2 expression in MCT cells, a cell model that shows significant upregulation of Col10a1 upon growth arrest. Both p63 and Runx2 showed around 2-fold upregulation in hypertrophic MCT cells. These data suggest that P63 may collaborate with Runx2 and together regulate Col10a1 expression during chondrocyte maturation. To further explore the putative function of P63 during skeletal development, we have successfully established transgenic mouse lines in which HA and Flag tagged human TAP63α cDNA (the longest P63 isotype) is driven by previously described hypertrophic chondrocyte-specific Col10a1 control element (Col10a1-TAP63α). By phenotypic analysis of these transgenic mice during different developmental stages, we will be able to define the consequence and the potential mechanism of P63 on skeletal development and skeletal disorders.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4972.
Abstract 4974: Myc-mediated suppression of the AU-binding protein Tristetraprolin controls Vegf expression
Authors: Rounbehler Robert J. , Li Weimin , Fallahi Mohammad , Cleveland John L.
Published: February 2011

The c-Myc oncoprotein is a transcription factor that drives cell proliferation, apoptosis, and angiogenesis during tumor development. Myc binds to target genes harboring preferred E-boxes to induce their expression, but this response only explains a fraction of the biological responses controlled by Myc. In human Burkitt lymphoma and the B cells and lymphomas of Eµ-Myc transgenic mice, a mouse model of Burkitt lymphoma (BL), Myc induces the expression of Vegf mRNA. Surprisingly, we found that Myc does not bind to the E-boxes found within the Vegf promoter, but rather induces this cytokine by stabilizing its Vegf transcripts. One mechanism that controls the stabilization of Vegf mRNA is the binding of AU-binding proteins (AUBPs) to AU-rich elements (AREs) found in the 3′ untranslated region (3′ UTR) of Vegf mRNA. AREs are present in the 3′ UTRs of approximately 10% of all human genes, including those encoding most of the angiogenic cytokines. Strikingly, expression profiling revealed that Myc regulates ∼30% of ARE-containing genes in human BL and in Eμ-Myc lymphomas. Interestingly, the expression of several AUBPs is altered in premalignant Eμ-Myc B cells, and Myc directly suppresses the expression of the RNA-destabilizing factor Tristetraprolin (TTP). These findings therefore suggest that Myc regulates Vegf expression indirectly, by down-regulating TTP. To directly test the importance of Myc-mediated repression of TTP, we developed Eμ-TTP transgenic mice, to enforce the expression of TTP in the B cells of Eμ-Myc mice. We predict uncoupling Myc's ability to repress TTP will delay tumor development by impairing Vegf expression and thus blocking the angiogenic response. These data provide a novel mechanism whereby Myc indirectly regulates hundreds of ARE-containing genes through the regulation of AUBPs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4974.
Abstract 4975: Dynamic in vivo interactions of the eleven-nineteen lysine-rich leukemia protein (ELL) in the mammalian nucleus
Authors: Wakano Clay , Fufa Temesgen D. , Stasevich Timothy J. , Avery Taara A. , McNally James G. , Gardner Kevin
Published: February 2011

The eleven-nineteen lysine-rich leukemia protein (ELL) is an elongation factor that increases the processivity of RNA polymerase II during transcription. ELL forms a fusion protein with the histone-methyltransferase mixed lineage leukemia protein (MLL) via a chromosomal translocation t(11;19)(q23;p13.1), often associated with acute myeloid leukemia (AML) in both adults and children. This chromosomal translocation alters both proteins, eliminating the carboxyl-terminal methyltransferase domain of MLL and the amino-terminal elongation inhibitory domain of ELL. The biological and regulatory role of normal ELL and ELL in the MLL-ELL fusion protein during transcription is not understood. Transcription is a multi-step process that can be separated into a series of events which involve proteins that form transient and stable complexes with the DNA template at various times during the transcription process. Using the Fluorescence Recovery After Photobleaching (FRAP) approach allows for the dissection of these events in transcription. Transcription in living cells has been examined by photobleaching fluorescently tagged RNA polymerase II, revealing its binding kinetics and quantifying the residency times associated with the different phases of transcription, with shorter residency times correlating with recruitment and initiation and longer residency times with elongation. We have used FRAP to examine the dynamic kinetics of ELL and pol II and other transcriptional elongation factors, including P-TEFb during active transcription in living cells in the presence or absence of specific inhibitors of transcription elongation and initiation. Our results suggest that there is a hierarchy of interactions between elongation factors and pol II during the transcription cycle that is orchestrated through specific interactions with ELL and gives general insights into how disruption of ELL function through its fusion to MLL may selectively influence transcriptional elongation events and gene expression in rapidly growing cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4975.
Abstract 4982: N-myc downstream-regulated gene 1/Cap43 expression promotes cell differentiation of human osteosarcoma cells
Authors: Fukushima Nobuhiro , Zenmyo Michihisa , Hiraoka Koji , Shoda Takanori , Matsugaki Toru , Komiya Setsuro , Ono Mayumi , Kuwano Michihiko , Nagata Kensei
Published: February 2011

N-myc downstream regulated gene 1 (NDRG1)/Cap43 is closely associated with cell differentiation, and its expression is induced by hypoxia and increasing intracellular calcium. Whether NDRG1/Cap43 expression in cancer cells is a predictive marker of good or poor prognosis in patients depends upon tumor types and differentiation status. In this study, we examined whether NDRG1/Cap43 expression was involved in differentiation of osteosarcoma cells, and used three osteosarcoma cell lines, MG63, U2OS, and SaOS2. Its expression in MG63 and U2OS was significantly enhanced by vitamin D3 that also induced production of osteocalcin, a differentiation marker of osteoblasts. Knockdown of NDRG1/Cap43 using siRNA also suppressed the production of osteocalcin and enhanced cell proliferation, accompanying by suppression of p21 expression. Furthermore, acquired invasiveness of osteosarcoma cells during invasion in Matrigel resulted in decreasing expression of NDRG1/Cap43. A possible role of NDRG1/Cap43 in cellular capacity for differentiation and invasion in osteosarcoma cells will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4982.
Abstract 4984: Androgen response element of the glycine N-methyltransferase gene is located in first intron
Authors: Lee Cheng-Ming , Yen Chia-Hung , Chen Yi-Ming A.
Published: February 2011

BACKGROUND. The prostrate cancer (PCa) susceptibility gene glycine N-methyltransferase (GNMT) is associated with the metastasis potential of PCa cells. Androgens, which participate in transcriptional regulation via an androgen receptor (AR), play an important role in PCa development. Our two goals were to determine whether GNMT is regulated by androgen, and to map androgen response elements (AREs).

METHODS. Real-time PCR was used to evaluate GNMT mRNA expression levels after LNCaP and DU145 cells were treated with R1881 (a synthetic AR agonist). Putative AREs were identified and tested using the MatInspector program and a luciferase reporter assay respectively. Electrophoretic mobility shift assay and chromatin immunoprecipitation were used to verify the binding properties of the GNMT-ARE motif.

RESULTS. Results from real-time PCR analyses indicate that R1881 is capable of inducing significant GNMT mRNA levels in AR-positive LNCaP cells, but not in AR-negative DU145 cells. According to in-silico screening prediction data, putative ARE1, ARE2 and ARE3 are located within GNMT intron 1, and ARE4 is located within intron 2. According to results from a luciferase reporter assay, only ARE3 mediated the transcriptional activation effects of R1881. Results from an electrophoretic mobility shift assay show that AR binds to ARE3 in vitro. Chromatin immunoprecipitation assay results indicate AR interaction with the ARE3-containing region in vivo.

CONCLUSION. Our data show that GNMT is an AR target gene, with its functional ARE located at +1114/+1128 in GNMT intron 1. We believe this information is valuable for clarifying the relationship between androgen and GNMT gene expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4984.
Abstract 4986: Urokinase plasminogen activator (uPA) interacts with nuclear HOXA5 and inhibits p53 expression in glioma stem cells
Authors: Gondi Christopher S. , Gorantla Bharathi , Fassett Daniel , Klopfenstein Jeffrey D. , Dinh Dzung H. , Gujrati Meena , Rao Jasti S.
Published: February 2011

Urokinase-type plasminogen activator (uPA) participates in diverse physiological processes, including glioma progression. HOXA5 is a known regulator of p53 expression, and overexpression of HOXA5 has been shown to increase p53 expression. In this study, we have demonstrated that uPA not only translocates to the nucleus but also interacts with the transcription factor HOXA5. We used CD133- and CD44-enriched U87 human glioma cells and 4910 human glioma xenograft cells, which exhibit cancer stem cell properties. Nuclear localization of uPA was determined by western blot analysis of nuclear fractions and by immunocytochemistry. Glioma stem cells showed increased nuclear localization of uPA as compared to normal glioma cells. To determine the in vitro interaction of uPA with HOXA5, we initially used membrane immobilized transcription factors incubated with purified uPA protein and looked for strong protein-protein interactions. We observed that uPA binds preferentially to HOXA5 transcription factor. Subsequently, we immunoprecipitated uPA or HOXA5 from nuclear fractions of U87 and 4910 normal and stem cells and tested for co-precipitation of HOXA5 or uPA respectively. Again, the results showed that uPA binds preferentially to nuclear HOXA5. To determine the effect of uPA or HOXA5 downregulation on these glioma cells, we used RNAi-expressing plasmids targeting uPA or HOXA5. The results showed that downregulation of uPA alone induced overexpression of p53 in both normal and glioma stem cells. Glioma stem cells downregulated for uPA showed at least a 5-fold increase in p53 expression when compared to uPA downregulated normal glioma cells. Downregulation of HOXA5 alone did not show significant change in expression levels of p53; however, simultaneous downregulation of uPA and HOXA5 did not show change in expression levels of p53. To determine whether uPA interacts directly with HOXA5 binding sequences, we performed an electromobility shift assay (EMSA). We observed that uPA does not bind to HOXA5 sequences but rather forms a complex with HOXA5 bound to DNA. In vivo studies showed that subcutaneous tumors injected intratumorally with siRNA-expressing plasmids targeting uPA or HOXA5 either singly or simultaneously showed strong p53 expression in uPA downregulated tumors with no significant change observed in other treatment conditions. Taken together, these results demonstrate that uPA behaves as a negative regulator of p53 expression in glioma cells.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4986.