bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3056: High levels of Wee1 are associated with malignancy and has potential as a therapeutic target in malignant melanoma
Authors: Magnussen Gry I. , Emilsen Elisabeth , Rosnes Anne K. , Flørenes Vivi Ann
Published: August 2012

Malignant melanoma is the most increasing form of cancer worldwide, in addition to being the second most common cancer in young adults. Whereas the prognosis is good when detected early, there are no curative treatments once the cancer has spread (stage IV). Thus, there is a precarious need for new and more effective treatment options. Since the G1/S checkpoint of the cell cycle is dysfunctional in the majority of human cancers, the G2/M transition has been a focus of attention for targeted therapy the recent years. The Wee1 kinase is a major regulator of the G2/M checkpoint, and may halt the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in the progression of malignant melanoma we examined its expression in a panel of paraffin-imbedded patient derived tissues of benign nevi, primary- and metastatic melanoma. We found that Wee1 expression increased in the direction of malignancy, and had a strong, positive correlation with known markers of proliferation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p<0.001), p21Cip1/WAF1 (p=00.3), p53 (p<0.025), thicker primary tumours (p=0.001), ulceration (p=0.005) and poor disease-free survival (p=0.008). Transfections using siWee1 in three metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumour promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed reduced viability (MTS analysis), DNA damage (γ-H2AX) and induction of apoptosis (DNA fragmentation, caspase 3- and PARP-cleavages), in the absence of Wee1. Thus, the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Moreover, our preliminary results have demonstrated that combined inhibition of Wee1 using (Wee1 inhibitor II/681641) and MAPK/ERK signalling (PD98059) further enhanced the therapeutic effect, as compared to Wee1 silencing alone. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas and indicate that targeting Wee1 alone or in combination with other targeted therapies or conventional radio- or chemotherapy may be a promising approach in the treatment of melanoma patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3056. doi:1538-7445.AM2012-3056
Abstract 3057: CDK5 modulates the cell cycle, apoptosis and paclitaxel sensitivity in human ovarian cancer cells with wild-type p53 function
Authors: Zhang Shu , Mao Weiqun , Ahmed Ahmed A. , Li Zong-Fang , Le Xiao-Feng , Bast Robert C.
Published: August 2012

Cyclin-dependent kinase 5 (CDK5) is a ubiquitously expressed serine/threonine kinase that was initially identified in bovine brain extracts and plays an important role in normal neuron development and neurodegenerative diseases. This molecule has recently been shown to possess many cellular functions in non-neuronal tissues as well. Using a kinome siRNA screen, we identified CDK5 as a kinase that modulates paclitaxel sensitivity in human ovarian cancer cells. The aim of this report is to identify mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancer cells. Despite nucleotide sequence homology with human CDK1, a role of CDK5 in regulating the cell cycle remains unclear. Here we demonstrate dual roles of CDK5 in regulating the cell cycle and apoptosis. We have found that knockdown of CDK5 with multiple siRNAs markedly inhibits cell proliferation and increases paclitaxel sensitivity in several p53 wild type ovarian cancer cell lines using assays for cell viability and clonogenic growth. CDK5 knockdown with or without paclitaxel treatment induces G1 arrest of the cell cycle and apoptotic cell death linked to a significant induction of p53, p21Cip1 and p27Kip1 proteins. Induction of G1 arrest following CDK5 knockdown depends on induction of p21Cip1 and p27Kip1, since silencing p21Cip1 and/or p27Kip1 dramatically reduces CDK5 knockdown-induced G1 arrest. A CDK5 knockdown-induced increase of p53 expression contributes both to CDK5 knockdown-induced G1 arrest and to apoptosis, as silencing p53 markedly diminishes CDK5 knockdown-induced G1 arrest and apoptosis. As expected, a CDK5 knockdown-induced increase in p21Cip1 expression depends on p53-mediated transcriptional activation demonstrated by QRT-PCR and reporter luciferase assays. CDK5 knockdown activates caspase-3 and a pan-caspase inhibitor completely blocks CDK5 knockdown-induced apoptosis. Significant Inhibition of Bcl-2 expression is found in CDK5 knockdown-induced apoptosis and additional inhibition of Bcl-2 expression is found with combined CDK5 and paclitaxel treatment. Taken together, these data suggest that CDK5 can modulate paclitaxel sensitivity through regulating both the cell cycle and apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells with wild-type p53 function, providing a novel approach for enhancing the efficacies of paclitaxel-based regimens for low grade ovarian cancer in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3057. doi:1538-7445.AM2012-3057
Abstract 3058: Mad1 Serine-214 phosphorylation is essential for the spindle assembly checkpoint
Authors: Yang Chunying , Guo Xiaojing , Hao Jianwei , Wang Haibo , zhang wei , xu Bo
Published: August 2012

The spindle assembly checkpoint (SAC), which blocks anaphase onset until all chromosomes have bioriented, is one of the key self-monitoring systems of the eukaryotic cell cycle. The mitotic arrest-deficient protein 1 (Mad1), a critical component of the SAC, is hyperphosphorylated in mitosis. However, the kinases responsible for Mad1 phosphorylation and its functional significance are not fully understood. Here we report that Mad1 is phosphorylated on Serine-214 in vivo and in vitro by the ATM kinase, a critical DNA damage response protein also activated in mitosis and required for the SAC. This phosphorylation facilitates the homodimerization of Mad1 and heterodimerzation with Mad2. Cells expressing the mutation of Serine-214 to Alanine show a defective SAC and shorter mitotic timing. Introduction of the Serine-214 to glutamic acid mutant Mad1 partially complements the SAC defect in ATM-deficient cells. Immunofluorescence experiments show that Mad1-Ser214 phosphorylation has a similar localization pattern to ATM Serine-1403 phosphorylation during mitosis. In clinical samples, we find that Mad1 Serine-214 phosphorylation is highly expressed in malignant breast cancer tissues and is significantly correlated with the mitotic index (p<0.001, Pearson Correlation Test), indicating the physiological significance of Mad1 Serine-214 phosphorylation. Together, these findings reveal an important role of ATM-mediated Mad1 phosphorylation in mitosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3058. doi:1538-7445.AM2012-3058
Abstract 306: TWEAK functions as chemotactic factor for glioma cells via Lyn activation
Authors: Dhruv Harshil D. , Jameson Nathan , Patel Falak , Nakada Mitsutoshi , Armstrong Brock , Winkles Jeffrey A. , Berens Michael E. , Tran Nhan L.
Published: August 2012

The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK using the Modified Boyden chamber assay. Because TWEAK has been shown to be expressed in normal astrocytes, microglia, and endothelial cells, we neutralized TWEAK using a recombinant soluble murine Fn14-Fc decoy receptor and examined the effect on glioma cell invasion in the ex vivo rat brain slice model. Incubation of the brain slice with the Fn14-Fc decoy receptor reduced glioma cell invasion as compared to control IgG treated brain slice. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of Src as detected by Western blot analysis using a pan-Src phosphorylation antibody. To determine which SFK member is phosphorylated by TWEAK, we applied the multiplex Luminex assay and identified Lyn as a candidate SFK. Immunoprecipitation of Lyn from TWEAK-treated cells verified that Lyn is phosphorylated as determined by phosphotyrosine immunoblot analysis. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 306. doi:1538-7445.AM2012-306
Abstract 3061: Colocalization of NQO1 with the mitotic spindle in human cells
Authors: Siegel David , Kepa Jadwiga K. , Ross David
Published: August 2012

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that catalyzes the two-electron reduction of quinones. NQO1 has also been shown to reduce superoxide to hydrogen peroxide and regulate ubiquitin independent 20S proteasome degradation of proteins such as p53. In normal tissues NQO1 is expressed primarily in epithelium, endothelium and adipocytes where it is believed to play a protective role. NQO1 is also expressed at relatively high levels in many human solid tumors where it has been targeted for its ability to bioactivate anti-tumor quinones. Interestingly, immunohistochemical staining for NQO1 expression in formalin-fixed human non-small cell lung cancer tissue sections revealed strong staining for NQO1 on the mitotic spindles of cells in mitosis. Immunocytochemical studies in combination with confocal microscopy using human nontransformed and transformed cells (HUVEC, HBMEC, astrocytes) as well as human cancer cell lines confirmed NQO1 staining of mitotic spindles. Staining of mitotic spindles for NQO1 was not observed in cells genotyped as homozygous for the NQO1*2 polymorphism or in cells pretreated with siRNA directed against NQO1. Double-labeling experiments using anti-alpha-tubulin and anti-NQO1 antibodies demonstrated colocalization of NQO1 with spindle microtubules. Pretreatment with NQO1 inhibitors (ES936 or dicumarol) did not prevent binding of NQO1 to mitotic spindles suggesting that binding is independent of the redox state of the enzyme. In cells not undergoing mitosis there was no apparent association of NQO1 with the cytoskeleton suggesting that binding of NQO1 to microtubules may be specific for mitotic spindles. Previous immunocytochemical studies by other investigators have shown that p53 and 20S proteasome could also be found in association with the mitotic spindle. Whether NQO1 binds directly to spindle microtubules or the binding of NQO1 to the spindle is dependent upon the association with other proteins such as p53 or 20S proteasome is currently under investigation (Supported by NIH grant R01ES018943).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3061. doi:1538-7445.AM2012-3061
Abstract 3064: An alternative mechanism of action for paclitaxel in breast cancer
Authors: Zasadil Lauren M. , Weaver Beth A.
Published: August 2012

Paclitaxel is a microtubule-stabilizing drug that binds to the β-tubulin subunit and accumulates in mitotic cells. As a chemotherapeutic agent, paclitaxel is used for the treatment of a variety of cancers including breast. Currently, no biomarker is available to predict which patients will benefit from paclitaxel treatment. Paclitaxel studies in cells focus primarily on relatively high concentrations of the drug, which cause cells to die following long-term mitotic arrest. The clinically relevant concentration of paclitaxel is currently unknown, although it is widely speculated to be in the low nM range. Cells exposed to low concentrations of paclitaxel complete mitosis with minimal delay, but frequently contain multipolar spindles. In chromosomally stable breast cancer cells, low concentrations of paclitaxel are sufficient to increase aneuploidy within two days of exposure. These data suggest that the mechanism of cell death due to low dose paclitaxel is likely to be quite different from the mechanism of cell death caused by high dose paclitaxel. They also predict that chromosomally instable (CIN) cells may be more sensitive to cell death caused by low doses of paclitaxel. CIN cells have an existing level of chromosomal missegregation, which is likely to be exacerbated by treatment with low doses of paclitaxel. Since missegregation of large numbers of chromosomes per division causes rapid cell death, these cells may be more likely to die after low dose paclitaxel treatment as a result of losing both copies of a vital chromosome. Chromosomally stable cells, which do not normally missegregate chromosomes with each division, would be more resistant to death following paclitaxel treatment than CIN cells. These data suggest that CIN may be used as a biomarker to determine which tumors are most likely to be sensitive to treatment with clinically relevant concentrations of paclitaxel.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3064. doi:1538-7445.AM2012-3064
Abstract 3065: Interferon consensus sequence-binding protein (ICSBP) regulates the transforming growth factor-beta type I receptor expression in osteosarcoma cells
Authors: Sung Jee Young , Kim Yong-Nyun , Park Byung-Kiu
Published: August 2012

Transforming growth factor-beta (TGF-β), known as a tumor suppressor, may exert tumor promoting activity in the advanced stage of cancer. Previously, we reported that interferon consensus sequence-binding protein (ICSBP), also known as interferon regulatory factor-8, up-regulates TGF-βtype I receptor (TGF-βR I), and augments growth in HL-60 leukemic cells. In the current study, we demonstrate that ICSBP expression up-regulates TGF-βR I and increase in cell growth in U2OS human osteosarcoma cells. To further investigate the mechanism of TGF-βR I up-regulation by ICSBP, we tested the possibility that ICSBP regulates promoter activity of TGF-βR I gene. We analyzed the promoter region of the human TGF-βR I gene in U2OS cells to find putative transcription elements regulated by ICSBP. Luciferase reporter gene assays indicated that ICSBP expression could enhance TGF-βR I gene expression, and by promoter deletion analysis we identified a functional ICSBP-dependent region located at nucleotides −300 to −1 in the TGF-βR I promoter. We also performed chromatin immunoprecipitation using anti-ICSBP and found that there are putative ICSBP binding sites in −300 to −1 region. In conclusion, ICSBP increased TGF-βR I expression by binding to TGF-βR I promoter (−300/−1) in U2OS cells. Exploration for the specific ICSBP binding sequences within TGF-βR I promoter (−300/−1) is under way, which may be helpful in understanding the regulatory mechanism involved in TGF-βR I expression by ICSBP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3065. doi:1538-7445.AM2012-3065
Abstract 3129: XPC protein attenuation and deficiency in tobacco smoke-induced DNA hypermethylation of tumor suppressor genes
Authors: Wang Gan , Xu Xiaoxin S. , Wang Le
Published: August 2012

DNA hypermethylation of tumor suppressor genes is a hallmark for many types of cancer, including lung cancer. The mechanism that leads to hypermethylation of these tumor suppressor genes in tumor cells is not fully understood. Importantly lung cancer is strongly associated with environmental factors, especially cigarette smoking. DNA damage generated by environmental carcinogens and their metabolites is believed to play an important role in lung cancer development. XPC is a DNA damage recognition protein required for nucleotide excision repair (NER) and many other DNA damage responses (e.g. checkpoint regulation and apoptosis). Attenuated levels of XPC protein have been detected in a majority of lung cancer patients. We studied the role of XPC protein attenuation and deficiency in cigarette smoke-caused DNA hypermethylation. Using primary human normal fibroblast (NF) and XPC-deficient human fibroblasts (XPC−), our DNA methylation studies revealed that the XPC deficiency caused greatly elevated levels of DNA hypermethylation in Brca1, Mlh1, and Xpc genes after exposure to tobacco smoke condensate (TSC). Our chromatin immunoprecipitation (ChIP) results indicated that the XPC deficiency led to a more increased binding of DNA methyltransferase 3A (DNMT3A) at the promoters of these genes following the TSC treatment; however, this binding was partially diminished when the XPC cells were treated with caffeine prior to the TSC treatment. The results of our IP studies further revealed that the ATR protein interacted with DNMT3A. In addition, our western blots revealed that the level of ATR protein phosphorylation at serine 428 was greatly increased in XPC cells after the TSC treatment. These results suggest that the XPC protein attenuation and deficiency may cause lung cancer as a result of tobacco smoking through activation of the ATR protein, which enhances the binding of DNMT3A at selected target sequences, resulting in hypermethylation of important tumor suppressor genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3129. doi:1538-7445.AM2012-3129
Abstract 3133: Regulatory sequences of the ATP-Binding Cassette Transporter A1 gene are hypermethylated in prostate cancer resulting in loss of gene expression
Authors: Lee Byron H. , Robinet Peggy , Smith Jonathan , Magi-Galluzzi Cristina , Klein Eric A. , Ting Angela H.
Published: August 2012

Introduction/Objective: Cholesterol homeostasis is altered in prostate cancer resulting in increased levels of cholesteryl esters and free cholesterol, but the mechanism behind these perturbations is not complete understood. We examine the role of DNA methylation in the regulation of ABCA1 expression. Methods: Genome wide methylation analysis using MBD-isolated Genome Sequencing (MiGS) was performed on the normal prostate epithelial cell line PrEC, the prostate cancer cell lines LNCaP and DU 145, 3 benign prostate specimens obtained from radical cystoprostatectomy, and 6 prostate cancer specimens obtained from radical prostatectomy. Bisulfite genomic sequencing was performed on the ABCA1 CpG island in PrEC and LNCaP to verify the methylation patterns discovered by MiGS. Real time RT-PCR was performed to assess ABCA1 mRNA levels in PrEC and LNCaP. LNCaP was treated with 5-aza-2′-deoxycytidine (5-aza), the LXR agonist T0901317, or a combination of the two drugs; and ABCA1 CpG island methylation was assessed by methylation-specific PCR (MSP) and gene expression was assessed by real time RT-PCR. Results: MiGS analysis revealed that ABCA1 CpG island methylation was present in LNCaP and 6 prostate cancer specimens but not PrEC, DU 145, or 3 benign prostate specimens. Bisulfite genomic sequencing primers were used to amplify fragments from the CpG island spanning −404 to +1,301 of the ABCA1 gene. LNCaP showed dense methylation in the entire region while PrEC showed only minimal methylation from −236 to −404. Real time RT-PCR revealed that there was 10-fold more ABCA1 mRNA in PrEC when compared with LNCaP. Treatment of LNCaP with 10µM T0901317 for 24 hours increased ABCA1 mRNA to 14 fold above untreated LNCaP without detectable changes in DNA methylation. Treatment with 5µM 5-aza for 1 week increased ABCA1 mRNA to 50 fold above control and resulted in loss of DNA methylation in the ABCA1 CpG island as detected by MSP. The combination of 5µM 5-aza and 10µM T0901317 increased ABCA1 mRNA expression in LNCaP to 320 fold above control. Conclusions: ABCA1 is hypermethylated in a subset of prostate cancers, but not normal prostate, resulting in loss of gene expression. Therapeutic strategies aimed at reducing intracellular cholesterol content solely by inhibiting HMG-CoA reductase may be hindered by this acquired deficiency in cholesterol export.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3133. doi:1538-7445.AM2012-3133
Abstract 3135: Epigenetic regulation of miR-1275 through histone H3 lysine 27 trimethylation during human glioma stem-like cell differentiation
Authors: Katsushima Keisuke , Shinjo Keiko , Ohka Fumiharu , Fujii Makiko , Osada Hirotaka , Sekido Yoshitaka , Natsume Atsushi , Kondo Yutaka
Published: August 2012

Functional and morphological heterogeneity characterizes aggressive glioblastoma and contributes to invasion, metastasis and drug resistance. The molecular mechanisms underlying this heterogeneity are poorly understood. Recent studies have revealed that tumors contain a minor population of tumor-initiating cells, called cancer stem cells (CSCs). The CSCs are considered able to aberrantly differentiate into diverse cell types. Such stem-like cancer cells have been well characterized in glioblastoma multiforme, and are referred to as glioma stem-like cells (GSCs). Using GSCs as a model, we investigated the roles of micro-RNA (miR) in GSCs differentiation. miR-microarray showed that 19 and 48 miRs were commonly upregulated and downregulated in two GSC lines during the differentiation, respectively. Among those miRs, quantitative-PCR analysis showed that miR-1275 was consistently downregulated during the GSC differentiation, while the expression of its target, oligodendrocyte specific protein (OSP/CLDN11), a marker of oligodendrocyte, was upregulated. Compellingly, inhibition of miR-1275 using specific antisense oligonucleotide increased the expression of OSP in GSC. Epigenetic analysis revealed that level of histone H3 lysine 27 trimethylation(H3K27me3) was enriched in the promoter of pri-miR-1275 during the GSC differentiation. Treatment of 3-Dezaneplanocin-A, an inhibitor of H3K27me3, impaired the GSC differentiation in parallel with sustained miR-1275 expression. In general, astrocytic component is the most prevalent in glioblastomas, while a subset of cases also contain an oligodendroglial component in the tumors. Our results illuminated the epigenetic regulatory pathways of miR-1275 closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity formation of glioblastomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3135. doi:1538-7445.AM2012-3135
Abstract 3136: C-Myc-mediated transcriptional repression of HPP1 in colon cancer by recruitment of HDACs
Authors: Wang Jian , Abul Elahi , Hernandez Jonathan , Clark Whalen , Achille Alex , Hao Jihui , Seto Ed , Shibata David
Published: August 2012

Background: Epigenetic gene silencing plays a critical role in tumorigenesis. HPP1, a putative tumor suppressor gene is downregulated in a number of tumor types including colon cancer. As a mechanism of suppression, promoter hypermethylation of HPP1 is known to be important, however the contributions and mechanisms of histone deacetylation have not been described. Additionally, the c-myc oncogene also contributes to the suppression of HPP1 via promoter binding to a cognate E-box site. We sought to elucidate the role of histone deacetylation and the potential interplay with c-myc in the transcriptional regulation of HPP1. Methods: The HPP1 non-expressing cell lines, HCT-116 and DLD-1, were treated with the DNA methylation inhibitor, 5-aza-2′-deoxycitidine (5-Aza-DC) and/or histone deacetylation (HDAC) inhibitors, including sodium butyrate (SB), Vorinostat (SAHA), trichostatin A (TSA) and valproic acid (VPA). Knockdowns of c-Myc and HDAC-1, 2, 3 and 4 were performed using siRNA. Protein and mRNA expression of HPP1 were determined by Western Blot and RT-PCR respectively. Furthermore, Chromatin immunoprecipitation (ChIP) and ChIP-re-ChIP experiments were used to demonstrate the binding of c-Myc and HDACs to the HPP1 promoter. Co-IP assays were used to test the physical interaction between c-Myc and HDAC3. After knockdown of HDAC3 using siRNA in HCT116, ChIP was also used to test the activity of acetylated histone 4 (AcH4). Results: 5-Aza-DC as well as HDAC inhibitors, including SB, TSA, SAHA and VPA, resulted in the re-expression of HPP1 in HCT-116 and DLD-1 cells. Individual HDAC inhibitors administered in combination with 5-Aza-DC resulted in a synergistic effect on HPP1 re-expression. Concordantly, knockdown of HDAC2 and HDAC3 in HCT116 cells, and HDAC1 and HDAC3 in DLD-1 cells by siRNA significantly enhanced HPP1 expression. Co-IP, ChIP and double ChIP experiments confirmed binding of c-myc to the HPP1 promoter as a complex with dominant recruitment of HDAC3. By ChIP assay, the levels of AcH4 increased significantly in HCT116 after knockdown of HDAC3. Conclusions: We have demonstrated that HDAC-1, -2 and -3 contribute to the epigenetic regulation of HPP1. Moreover, c-myc binds to the HPP1 promoter and mediates epigenetic suppression via the dominant recruitment of HDAC3 with subsequent enhancement of histone deacetylation. Our findings may lead to a greater biologic understanding for the application of the targeted use of HDAC inhibitors for anti-cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3136. doi:1538-7445.AM2012-3136
Abstract 3137: Epigenetic regulators influencing dual histone reader TRIM24 in estrogen receptor-mediated transcription in breast cancer
Authors: Yiu Teresa T. , Barton Michelle C.
Published: August 2012

Nuclear receptor-mediated regulation of gene expression, including estrogen-receptor-alpha (ERα), relies on a complex network of proteins that impose either repression or activation of gene expression. Among these, epigenetic “reader” proteins recognize combinatorial PTMs on histones, through distinct structure domains such as tandem plant homeodomains (PHD) and bromodomains (Bromo). We recently reported that tripartite motif-containing 24 (TRIM24), by means of its C-terminal tandem PHD finger and Bromo domains, recognizes unmethylated histone H3 lysine K4 (H3K4me0) and acetylated histone H3 lysine K23 (H3K23ac) within the same histone tail. In the presence of estrogen, dual chromatin reader TRIM24 preferentially recognizes these two histone marks, binds to ERα and regulates estrogen-dependent target genes. Depletion of TRIM24 inhibits breast cancer cell growth and enhances tamoxifen-induced growth inhibition. In addition, overexpression of TRIM24 correlates with decreased overall survival in breast cancer patients. We believe that TRIM24 can be a potential biomarker for breast cancer development, as well as a target for therapeutic treatment. The goal of my research is to target TRIM24 in breast cancer by identifying upstream epigenetic regulators that may influence TRIM24's function in ERα-regulated transcriptional activation. For TRIM24-regulated ERα target genes, estrogen stimulation results in a global loss of H3K4me2 at estrogen-responsive elements (EREs), where TRIM24 and ERα are recruited. Global binding profile of TRIM24 by ChIP-seq also shows that TRIM24 preferentially binds to regions depleted of H3K4me2, suggesting that upstream regulators may determine TRIM24 binding to chromatin. Demethylation of histones is mediated by specific enzymes called histone demethylases (HDMs). When we increase H3K4me2 at EREs by inhibiting the enzymatic activity of HDM, recruitment of both TRIM24 and ERα to target genes is diminished. As a result, activation of TRIM24-regulated estrogen-responsive genes is impaired. Previous work has suggested that the presence of threonine T6 phosphorylation on histone H3 (H3T6ph) prevents HDMs from demethylating H3K4me2. H3T6ph may therefore impose an inhibitory mark for LSD1-mediated demethylation of H3K4me2, and serves as an added layer of regulation for TRIM24 binding to histones, affecting subsequent function of TRIM24 as an ERα coactivator. These results may give insights into future therapeutic studies of targeting TRIM24 in breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3137. doi:1538-7445.AM2012-3137
Abstract 3138: Epigenetic regulation of Src pathway in pancreatic adenocarcinomas
Authors: Padin-Iruegas Elena , Evgenyeva Elena , Herranz Michel , Abdulkader Ihab , Brozos Elena , Pombo Maria , Antúnez Jose Ramón , Ruibal Alvaro , López Rafael
Published: August 2012

Purpose: Src cascade is crucial in tumor growth and metastasis development. Nowadays, one of the most relevant mechanisms for gene control is epigenetic mechanisms, both methylation and acetylation. We study the epigenetic regulation of Src pathway in pancreatic adenocarcinomas. The main purpose of this study was to obtain translational results, we can apply in the clinical practice. Methods: 70 pancreatic adenocarcinomas and normal tissue from the same patient were analyzed. DNA from laser microdissected paraffin embedded sample was obtained, bisulfite treated, methylation specific PCR (MSP) amplified, and agarose visualized. Some cases were bisulfite sequenced for verification. Rassf1a, Cox2, Socs, E-Cadherin, DAP-kinase, TIMP3, TWIST1, p16 and Cyclin-D2 genes were analyzed due to their implication with Src pathway. All patients signed informed consent and detail clinic histories were reviewed. Results: We found gene hypermethylation in all the selected targets ranged between 23.5% (DAP-K) to 73% (TWIST1), i.e. RASSF1α (48.7%); p16 (38.2%)… Bisulfite promoter sequence confirms MSP results. Patient clinical profile clustered in some well defined groups. Epigenetic profile also showed very nice association. Combine clinical and epigenetic profile showed a specific and characteristic correlation between some clinical parameters and specific gene methylation profile. In this sense, for instance, we found a perfect match between just RASSF1α promoter hypermethylation with clinical parameters: pTNM and tumor localization. Conclusions: Gene hypermethylation control seems important in pancreatic adenocarcinoma behavior. Src pathway could be implicated in tumoral development, both directly and indirectly (through i.e. EGFR, HER2/Neu, Integrins and SPARC). Src pathway methylation control could be important in development and progression of pancreatic carcinoma (up to 73%). An “epigenetic fingerprint” of pancreatic adenocarcinoma was found from clinical and epigenetic clustering analysis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3138. doi:1538-7445.AM2012-3138
Abstract 3140: Constitutive and regulated expression of GPR109A by DNA methylation and IFN-γ in human colon carcinoma cells
Authors: Bardhan Kankana , Ganapathy Vadivel , Liu Kebin
Published: August 2012

Butyrate, one of the short chain fatty acids generated in the colon by microbiota from dietary fiber, has shown both preventative and therapeutic potential to counteract inflammatory disease and inflammation-mediated colorectal cancer. GPR109A, a recently identified receptor for butyrate, is highly expressed in normal colonic tissue, but is often down-regulated in human colon carcinoma cells. The molecular mechanisms underlying regulation of GPR109A expression in human colon carcinoma cells is unknown. Here we demonstrated that although there is no CpG island in the human GPR109A promoter region, the GPR109A promoter is hypermethylated in human colon carcinoma cell lines and in colon carcinoma tissue specimens. Interestingly, despite the hypermethylation of the promoter, IFN-γ, a cytokine secreted by activated cytotoxic T lymphocytes (CTL), effectively up-regulated GPR109A expression in human colon carcinoma cells, and this regulation is mediated at the transcriptional level. Furthermore, we demonstrated that the IFN-γ-activated pSTAT1 regulates GPR109A expression by directly binding to the Gamma Activation Site (GAS) elements in the GPR109A promoter region in human colon carcinoma cells. At the functional level, IFN-γ-mediated up-regulation of GPR109A resulted in increased sensitivity of the tumor cells to butyrate-induced cell death and increased level of histone acetylation in human colon carcinoma cells. In summary, our data demonstrated that although GPR109A can be constitutively silenced by DNA methylation in human colon carcinoma cells, tumor-infiltrating CTLs might use secretion of IFN-γ to up-regulate GPR109A as a mechanism to re-activate GPR109A, thereby restoring functional response of colon carcinoma cells to butyrate in the tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3140. doi:1538-7445.AM2012-3140
Abstract 3147: Differential epigenetic regulation of TOX subfamily high mobility group box genes in lung and breast cancers
Authors: Tessema Mathewos , Yingling Christin M. , Grimes Marcie J. , Thomas Cynthia L. , Liu Yushi , Leng Shuguang , Joste Nancy , Belinsky Steven
Published: August 2012

Aberrant cytosine methylation affects regulation of hundreds of genes during cancer development. In this study, a novel aberrantly hypermethylated CpG island in cancer was discovered within the TOX2 promoter. TOX2 was unmethylated in normal cells but 28% lung (n = 190) and 23% breast (n = 80) tumors were methylated. Expression of two novel TOX2 transcripts identified was significantly reduced in primary lung tumors than distant normal lung (p < 0.05). These transcripts were silenced in methylated lung and breast cancer cells and 5-Aza-2-deoxycytidine treatment re-expressed both. Extension of these assays to TOX, TOX3, and TOX4 genes that share similar genomic structure and protein homology with TOX2 revealed distinct methylation profiles by smoking status, histology, and cancer type. TOX was almost exclusively methylated in breast (43%) than lung (5%) cancer, whereas TOX3 was frequently methylated in lung (58%) than breast (30%) tumors. TOX4 was unmethylated in all samples and showed the highest expression in normal lung. Compared to TOX4, expression of TOX, TOX2 and TOX3 in normal lung was 25, 44, and 88% lower, respectively, supporting the premise that reduced promoter activity confers increased susceptibility to methylation during lung carcinogenesis. Genome-wide assays revealed that siRNA-mediated TOX2 knockdown modulated multiple pathways while TOX3 inactivation targeted neuronal development and function. Although these knockdowns did not result in further phenotypic changes of lung cancer cells in vitro, the impact on tissue remodeling, inflammatory response, and cell differentiation pathways suggest a potential role for TOX2 in modulating tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3147. doi:1538-7445.AM2012-3147
Abstract 3151: Molecular and clinicopathological impact of miR-375 deregulation in prostate cancer
Authors: Nolte Elke , Wach Sven , Stoehr Robert , Hartmann Arndt , Szczyrba Jaroslaw , Graesser Friedrich , Wieland Wolf , Wullich Bernd , Taubert Helge
Published: August 2012

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. As recently reported by our group, miR-375 was found to be upregulated in prostate cancer using deep sequencing and microarray analyses. We now set out to study whether there is an association between miR-375 expression and the expression of different gene members of the epithelial-mesenchymal transition (EMT) pathway. Material and Methods: Expression of miR-375 and genes of the EMT pathway (ZEB1, ZEB2, N-Cadherin) was measured by qRT-PCR in 26 pairs of prostate cancer and corresponding non-tumor prostate tissue. Bivariate correlation analyses of miR-375 and EMT members as well as for miR-375 and clinicopathological features were carried out. Results: MiR-375 showed a significant upregulation in prostate cancer using qRT-PCR (p=0.0019, paired student's t-test), whereas ZEB1, ZEB2, and N-Cadherin were consistently found downregulated (p<0.05, paired student's t-test). Applying bivariate correlation analysis, a negative correlation of miR-375 expression to N-Cadherin, ZEB1, and ZEB2 could be documented (p<0.05, Spearman Rho test). Furthermore, we could detect an inverse correlation of miR-375 expression to patient age (p=0.002, Spearman Rho test). Remarkably, miR-375 expression higher than median is significantly correlated to a lower tumor grade (p=0.011, Fisher's exact test). Conclusion: Expression of miR-375 is inversely correlated to the expression of N-Cadherin, ZEB1, and ZEB2. As SLUG is the only member of the EMT pathway, which is a predicted target of miR-375, we suppose SLUG to constitute the putative link between miR-375 and EMT. Furthermore, our findings show that expression of miR-375 is correlated with patient age and tumor grade.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3151. doi:1538-7445.AM2012-3151
Abstract 3152: Molecular networks regulated by tumor suppressive microRNA-1 and microRNA-133a in head and neck squamous cell carcinoma
Authors: Nohata Nijiro , Hanazawa Toyoyuki , Kinoshita Takashi , Kikkawa Naoko , Fuse Miki , Chiyomaru Takeshi , Yoshino Hirofumi , Enokida Hideki , Nakagawa Masayuki , Okamoto Yoshitaka , Seki Naohiko
Published: August 2012

(Background) Head and neck squamous cell carcinomas (HNSCC) represent 6% of all cancers. Despite of considerable advances in surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients is less than 40% in advanced stage. The understanding of the new molecular networks of HNSCC oncogenesis by recent genome analysis would be helpful in improving diagnosis and therapy of the disease. Our expression signatures of human cancer including HNSCC revealed that the expression of microRNA-1 (miR-1) and microRNA-133a (miR-133a) were significantly reduced in cancer cells. In human genome, miR-1 and miR-133a located same chromosomal regions (miR-1-2 and miR-133a-1 on 18q11.2, and miR-1-1 and miR-133a-2 on 20q13.33) called cluster. Previously, our group reported that miR-1 and miR-133a function as tumor suppressors in several types of cancers including HNSCC. In this study, we identify the novel molecular networks regulated by miR-1 and miR-133a commonly in HNSCC. (Methods) Genome-wide gene expression analysis was performed to identify the molecular networks of miR-1 and miR-133a by microarray technique. A luciferase reporter assay was used to identify the actual binding site between miR-1 and miR-133a and these candidate target genes. Cell proliferation, migration and invasion assays were performed to investigate the functional significance of target genes in HNSCC cell lines. (Results) Genome-wide molecular targets search and luciferase reporter assay showed that transgelin-2 (TAGLN2), prothymosin-alpha (PTMA) and purine nucleoside phosphorylase (PNP) were directly regulated by miR-1 and miR-133a commonly. Silencing of these genes studies demonstrated significant inhibition of cell proliferation, migration and invasion in HNSCC cells. (Conclusions) TAGLN2, PTMA and PNP were directly regulated by tumor suppressive miR-1 and miR-133a commonly. These genes may function as oncogenes contributed to cell proliferation, migration and invasion in HNSCC. Tumor suppressive miR-1 and miR-133a and target oncogenes may provide new insights into the mechanisms in cancer. Our findings have therapeutic implications and may be exploited for future HNSCC treatments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3152. doi:1538-7445.AM2012-3152
Abstract 3155: High-throughput profiling of miRNA expression in plasma and cerebrospinal fluid using OpenArray® platform for biomarker discovery
Authors: Liang Yu , Mouanoutoua Mokang , Shen Jianhe , Terashima Keita , Lau Ching
Published: August 2012

OpenArray® platform can be used for expression profiling using TaqMan® assays that enables higher throughput, less assay consumption, and lower sample input than other low-density assay platforms. One OpenArray® plate has 3,072 through-holes, each of which requires only 33nl of reaction volume. We combined the workflow of Megaplex™ RT primer pools and pre-amplification with TaqMan® MicroRNA assays on OpenArray® plates. Each OpenArray® plate provides the capacity of 3 samples, and each run can take up to 3 plates with a run time about 2.5 hours. Therefore, each typical work day can have 4 instrument runs generating expression data of 754 miRNAs from 36 samples. This platform is ideal to identify potential miRNA biomarkers within a short period of time by screening a large cohort of clinical specimens that might have limitations in sample size, contamination of PCR inhibitors, or low abundance of target miRNAs. We optimized the original workflow of Megaplex™ RT primer pools and pre-amplification before applying to the OpenArray® plates to ensure increasing detection sensitivity while maintaining the linearity in plasma samples. In a proof-of-principle investigation, we applied the new protocol to a small panel of plasma and cerebrospinal fluid (CSF) specimens derived from pediatric patients diagnosed with either germinoma or non-germinoma germ cell tumors of the brain, and benchmarked with another miRNA expression profiling platform, TaqMan® Array Card (TAC), and OpenArray® plates demonstrated good concordance with the TAC. Expression patterns of miRNAs detected in our CSF samples are largely consistent with previously published miRNAs differentially expressed in primary cancerous tissues between these two types of intracranial germ cell tumors. (For Research Use Only. Not for use in diagnostic procedures.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3155. doi:1538-7445.AM2012-3155
Abstract 3158: MicroRNA signatures associate with pathogenesis and progression of osteosarcoma
Authors: Salah Zaidoun , Jones Kevin B. , Mare Sara Del , Aqeilan Rami
Published: August 2012

Osteosarcoma is the most common sarcoma in the skeleton, but remains elusive with regard to molecular etiology. MicroRNAs (miRNAs) have demonstrated far-reaching effects on the cellular biology of development as well as oncogenesis. Here we identify an miRNA signature reflecting the pathogenesis of conventional (osteoblastic/fibroblastic) osteosarcoma from surgically procured samples from human patients. The signature includes high expression of miR-181a, miR-181b, and miR-181c as well as reduced expression of miR-29b, miR-16, and miR-142-5p. Osteosarcomas of varied histology subtype co-localized on unsupervised hierarchical clustering, suggesting that the signature profile relates to oncogenesis itself, rather than simply a differentiation phenotype. We also demonstrated that miR-181b and miR-29b exhibit restricted expression to distinct cell populations in the tumor tissue. Further, higher expression of miR-27a and miR-181c* in pre-treatment biopsy samples characterized patients who developed clinical metastatic disease. In addition, higher expression of miR-451 and miR-15b in pre-treatment samples correlated with subsequent positive response to chemotherapy. In vitro functional validation of miR-16 and miR-27a confirmed opposing roles in human osteoblast and osteosarcoma cell lines. Furthermore, predicted target genes for miR-16 and miR-27a were confirmed as down-regulated by real-time PCR. Affymetrix array profiling of cDNAs from osteosarcoma specimens and controls were interrogated according to predicted targets of miR-16, miR142-5p, miR-29b, miR-181a/b, and miR-27a. This analysis revealed positive and negative correlations highlighting pathways of known importance to osteosarcoma, as well as novel genes. Thus, our findings establish miRNA signatures associated with pathogenesis, metastasis, and responsiveness to treatment in osteosarcoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3158. doi:1538-7445.AM2012-3158