bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2147: The RRas2/TC21 GTPase is essential for breast tumorigenesis and lung metastasis
Authors: Larive Romain M. , Cañamero Marta , Bustelo Xosé R.
Published: August 2012

Rras2/TC21 is a Ras superfamily GTPase with still unknown functions in vivo. Several observations suggest that this protein may be involved in tumorigenic processes. Thus, it has been shown that the expression of constitutively active RRas2 mutants leads to high levels of cell transformation in vitro. Moreover, this GTPase has been found overexpressed in a number of tumors, including lymphomas, oral and esophageal carcinoma, and breast cancer. To address the role of this protein in epithelial tumors, we have carried out an extensive characterization of this GTPase using RRas2 knockout mice and shRNA knockdown tumor cells. These studies revealed that RRas2 is not important for the development and/or malignant progression of skin and oral squamous cell carcinoma. Instead, we have observed that the RRas2 deficiency severily impairs breast cancer tumorigenesis and the tropism of metastatic cancer cells to the lung. The characterization of the role of RRas2 in primary tumorigenesis indicates that this GTPase is important for the proliferation and angiogenesis of tumors. In the case of metastasis, RRas2 is important for the survival and proliferation of cancer cells in the lung parenchyma. By contrast, this GTPase is not required for the lymph node infiltration, intravasation, or extravasation of cancer cells. Interestingly, the requirement of RRas2 is still maintained in breast cancer cells that express active mutants of K-Ras, indicating that RRas2 plays essential roles downstream of both Her2 and K-Ras. Signaling experiments indicate that RRas2 contributes to those processes by modulating the activation of phosphatidylinositol 3-kinase ≤ and downstream targets such as Akt, FoxO1/FoxO3 and S6 ribosomal protein.

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 2147. doi:1538-7445.AM2012-2147
Abstract 215: Mechanisms of p53 activation by NIAM, nuclear interactor of ARF and MDM2
Authors: Reed Sara , Tompkins Van , Hagen Jussara , Thies Katie , Cryderman Diane , Wallrath Lori , Quelle Dawn
Published: August 2012

Nuclear interactor of ARF and Mdm2, NIAM, is a novel regulator of the ARF-Mdm2-p53 tumor suppressor pathway. It collaborates with ARF, promotes p53 transcriptional activity, and is negatively regulated by Mdm2-mediated ubiquitination and proteasome degradation. How NIAM activates p53 is not known, however its ability to do so in ARF-null cells reveals it is an ARF-independent process. Our preliminary data suggest that NIAM could activate p53 by either competing Mdm2 away from p53-Mdm2 complexes and/or by inducing lysine 120 acetylation of p53 via the Tip60 acetyltransferase. p53 acetylation at K120 is known to be regulated by two different histone acetyltransferases, Tip60 and MOF, and to promote the specific upregulation of apoptotic genes by p53. Our preliminary data show that NIAM interacts with Tip60 and strongly promotes p53 acetylation of K120, suggesting a functional NIAM-Tip60 interaction in vivo. Tip60 is an established chromatin modifying protein that influences chromatin structure and cancer gene expression. Consistent with these characteristics of Tip60, we discovered that NIAM is a chromatin-associated protein that binds to transcriptionally active regions of the genome. Other findings connect Tip60 and NIAM. Tip60 acts through p53, ARF and Myc to control transcription and promote the DNA damage checkpoint response. We found that NIAM, like Tip60, localizes to sites of DNA repair in response to DNA damage and it is already established that the absence of either protein leads to chromosomal instability. These compelling observations link NIAM with chromatin regulation and DNA damage repair, potentially via Tip60 signaling, all of which are important for the maintenance of chromosomal stability and prevention of 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 215. doi:1538-7445.AM2012-215
Abstract 2152: p38α MAPK regulates signal transduction between estrogen receptor (ER) and its antagonist by switching ER signaling from the classical to the non-classical pathway
Authors: Chen Guan , Zhi Huiying , Hou Songwang , Wang Phillip , Huang Jian , Basir Zainab , Chitambar Christopher R. , Qi Xiaomei
Published: August 2012

Estrogen receptor ≤ (ER) plays a decisive role in breast cancer growth through regulating gene expression via the classical {ERE (estrogen-response element) binding} and the non-classical (AP-1 recruitment by interacting with c-Jun) pathways and serves as the only target for hormone therapy. However, anti-estrogen acts on several cascades beside ER and the implications of these “off-target” effects on hormone sensitivity remain unexplored. Here we report that p38α MAPK is selectively activated by treatment of breast cancer cells with the antiestrogen tamoxifen (TAM) and activated p38α in turn switches ER signaling from the classical to the non-classical pathway leading to increased hormone sensitivity. This occurs primarily through p38α phosphorylating ER/S118 leading to its S118-dependent proteasome-degradation through the E3 ligase E6AP resulting in decreased ERE activity. Unexpectedly, Ser-118 is also required for ER to form a complex with p38α, for p38α to stimulate AP-1, to inhibit ERE, and to sensitize breast cancer cells to TAM-induced growth inhibition and ER/S118 phosphorylation alone is both necessary and sufficient for ER binding to the AP-1 DNA. These results indicate that Ser-118 phosphorylation serves as a central switch for p38α to regulate the ER signaling distribution, which may be targeted to increase breast cancer hormone sensitivity.

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 2152. doi:1538-7445.AM2012-2152
Abstract 2159: Targeting GRB7/ERK/FOXM1 signaling cascade is a promising therapeutic approach in ovarian cancer
Authors: Chan David W. , Ngan Hextan YS
Published: August 2012

Ovarian cancer is a higly mortal disease with poor prognosis and especially in high grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers remains unclear. In this study, we found that GRB7 (P < 0.0001), phospho-ERK1/2 (P < 0.0001) and FOXM1 (P = 0.001) were overexpressed and associated with high grade ovarian cancers by immunohistochemical analysis. Intriguingly, the expressions of GRB7, phospho-ERK and FOXM1 showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancer (P < 0.001). Biochemical studies using western blot analysis demonstrated that enforced expression of GRB7 could elevate the expressions of phospho-ERK and FOXM1, while enforced expression of FOXM1 could not alter levels of GRB7 and phospho-ERK. However, inhibition of ERK signaling by U0126 or PD98059 reduced the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or decreased FOXM1 expression by thiostrepton significantly inhibited cancer cell growth in vitro and in vivo. Collectively, our findings suggest that targeting GRB7/ERK/FOXM1 signaling cascade is a promising molecular therapeutic choice in combating ovarian 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 2159. doi:1538-7445.AM2012-2159
Abstract 2167: The tumor suppressor CDC73/parafibromin is required for the maintenance of histone 2B monoubiquitination both in vitro and in vivo
Authors: Hahn Michael A. , Dickson Kristie-Ann , Jackson Stuart , Clarkson Adele , Gill Anthony J. , Marsh Deborah J.
Published: August 2012

CDC73 (cell division cycle 73), also known as parafibromin, is a ubiquitously expressed tumor suppressor that is mutated in the germline of patients with the familial disorder Hyperparathyroidism Jaw Tumor syndrome, as well as somatically mutated in sporadic parathyroid cancer. Furthermore, CDC73 has been implicated in breast, gastric, renal and colorectal tumorigenesis. CDC73 is a member of the PAF1 (RNA polymerase II-associated factor 1) transcriptional complex (PAF1c), along with PAF1, CTR9, LEO1, and SKI8. Consistent with roles in transcription, CDC73 is localized to the nucleus, and has also been reported in the nucleolus. This tumor suppressor has been shown to function as a negative regulator of cell cycle progression and to promote apoptosis, as well as have a role in the regulation of 3′ processing of histone mRNA. We used CDC73 as bait in a yeast two-hybrid assay and identified the E3 ubiquitin ligase ring finger proteins RNF20 and RNF40 as CDC73 binding partners. RNF20 and RNF40 exist in a heterodimeric complex that functions to monoubiquitinate histone H2B at lysine 120 (H2B-K120). Monoubiquitination of H2B-K120 is associated with transcription in undamaged cells, is induced after DNA damage, and has a role in the maintenance of replication-dependent histone mRNA 3′ - end processing. We have confirmed the interaction of CDC73 with both RNF20 and RNF40 in mammalian cells by co-immunoprecipitation of overexpressed and endogenous proteins from human embryonic kidney (HEK293) cells. Down-regulation of endogenous CDC73 by siRNA in HEK293 cells and the breast cancer cell line MCF7 led to a significant reduction of monoubiquitinated H2B-K120. Furthermore, in a series of eleven parathyroid carcinomas harboring mutations in CDC73 that led to loss of nuclear CDC73, monoubiquitinated H2B-K120 was either absent or significantly reduced relative to benign parathyroid tumors that expressed nuclear CDC73. siRNA down-regulation of other PAF1c members in MCF7 cells also led to reduction in levels of monoubiquitinated H2B-K120. Given that CDC73 is the only PAF1c member in which cancer-associated mutations have been reported, the translational significance of these findings are unclear. Loss of the CTR9 locus has been reported in pancreatic cancer, as well as overexpression of PAF1. PAF1c members may have a greater role in malignancy than previously described. This is the first report to demonstrate pathogenic mutations affecting monoubiquitination of a histone. We propose that loss of H2B-K120 monoubiquitination is a major mechanism whereby CDC73 mutations exert their tumorigenic effect.

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 2167. doi:1538-7445.AM2012-2167
Abstract 2172: Anti-oncogenic role of peroxisome proliferator-activated receptor-α/β (PPARδ/α) involves inhibition of mitosis and regulation of Hras1-induced senescence
Authors: Zhu Bokai , Khozoie Combiz , Ferry Christina H. , Markell Lauren M. , Bility Moses T. , Blazanin Nicholas , Glick Adam B. , Gonzalez Frank J.
Published: August 2012

Ligand activation of PPARδ/α inhibits chemically-induced skin tumorigenesis and Pparα/α-null mice exhibit enhanced chemically-induced skin tumorigenesis compared to wild-type mice. The mechanism that mediates inhibition of skin carcinogenesis by PPARδ/α is still being unraveled and was examined in this study. Ligand activation of PPARδ/α caused a negative selection against cells expressing higher levels of HRAS by inducing a mitotic block. Mitosis-related genes that are predominantly regulated by E2F were induced to a higher level in HRAS-expressing Pparα/α-null keratinocytes as compared to HRAS-expressing wild-type keratinocytes. Ligand activated PPARδ/α repressed expression of these genes by direct binding with p130/p107, facilitating nuclear translocation, and increasing promoter recruitment of p130/p107. In addition, co-treatment with a PPARδ/α ligand and various mitosis inhibitors increases the efficacy of increasing G2/M arrest. PPARδ/α is also required for HRAS-induced senescence and suppression of malignant conversion of benign papillomas. HRAS-expressing Pparα/α-null keratinocytes evade senescence by utilizing the PI3K-AKT rather than the MEK-ERK pathway. PPARδ/α positively regulates MEK-ERK pathway by maintaining RASGRP1 levels and negatively regulating the PI3K-AKT pathway by repressing ILK and PDPK1 expression. Heightened AKT activity increases endoplasmic reticulum (ER) stress in Pparα/α-null keratinocytes, which in turn helps maintain higher AKT activity, leading to evasion of senescence. In addition, enhanced HRAS activity increases the synthesis of endogenous PPARδ/α ligands, thus providing a PPARδ/α-dependent cell-autonomous mechanism to suppress HRAS-induced carcinogenesis. (Supported by CA124533).

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 2172. doi:1538-7445.AM2012-2172
Abstract 2176: Deletion of 3p14 is linked to ERG-fusion positive prostate cancers and harbors the FOXP1 tumor suppressor gene
Authors: Simon Ronald , Krohn Antje , Burkhardt Lia , Bachmann Frederic , Schlomm Thorsten , Steuber Thomas , Gräfen Markus , Sauter Guido , Minner Sarah , Sirma Hüseyin
Published: August 2012

Genetic rearrangements involving 3p14 are frequent alterations in prostate cancer, affecting approximately 15-20% of tumors. Both the clinical significance and the tumor suppressor at 3p14 locus are currently unknown. We performed SNP array analysis of 72 primary prostate cancers and show a commonly deleted region at 3p14 in 14 (19%) of the tumors. Our data indicate the existence of a small, recurrent deletion in a significant fraction of prostate cancers harboring 8 genes only. Ten of 14 tumors with 3p14 deletions detected in our study included the FOXP1 locus. To estimate the prevalence and clinical significance of FOXP1 deletions, we analyzed a tissue microarray containing 4,699 prostate cancers with histopathological and clinical follow-up data. Heterozygous FOXP1 deletions were found in 15% of tumors, and were strongly associated with ERG-fusion positive cancers (p<0.0001). No homozygous deletions were detected. FOXP1 deletions were unrelated to tumor stage or Gleason grade. FOXP1 deletions were linked to early PSA recurrence in ERG-positive cancers (p=0.0576) but not in ERG negative tumors (p=0.5425). To better understand the association between ERG fusion and FOXP1 expression, we performed FOXP1 immunohistochemistry on our TMA. FOXP1 expression was found in virtually all (95%) prostate cancers, and was particularly strong in ERG-fusion positive tumors. This finding argues for an important role of FOXP1 in prostate cancer in general and for ERG fusion positive tumors in particular. Since FOXP1 is a transcriptional repressor, we hypothesized that upregulation might be a consequence of ERG activation in order to control transcription of ERG target genes. However, neither expression of ERG in LNCaP cells, or depletion of ERG in VCaP cells had an effect on FOXP1 mRNA levels, strongly arguing against a direct link between ERG and FOXP1 at least in the tested cell lines. In addition neither the transcription nor the steady state levels of FOXP1 protein were modulated by activation of AR signaling. Finally, colony formation assays in both benign and malignant prostate cell lines revealed that FOXP1 may act as a tumor suppressor. In summary, our study demonstrates that FOXP1 functions as one of the tumor suppressor genes in prostate cancer that is targeted by genomic deletions at 3p14.

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 2176. doi:1538-7445.AM2012-2176
Abstract 2183: Determining the functional roles of the specific isoforms of ARID3B from cellular localizations
Authors: Deneke Victoria E.
Published: August 2012

Ovarian cancer is the leading cause of death from a gynecological malignancy in the United States, yet the genetic mechanisms that contribute to ovarian cancer progression are poorly understood. We have determined that ARID3B, a member of the AT-rich interaction domain (ARID) family of proteins, is overexpressed in ovarian cancer. Therefore, we want to assess if ARID3B plays an important role in ovarian cancer progression. We have demonstrated that there are two ARID3B isoforms: the long form (ARID3BFL at 61kDa) and the short form (ARID3Bsh at 28kDa). We show that the ARID3BFL is mostly found in the nucleus and some is found in the membrane of cells. Furthermore, the short isoform of ARID3B (when overexpressed) is primarily found in cytoplasmic and membrane/mitochondria fractions. Therefore, we hypothesize that by virtue of these unique cellular distributions that ARID3B isoforms elicit different cellular responses. ARID3BFL binds DNA and induces apoptosis in ovarian cancer cell lines while ARID3Bsh does not induce cell death. Further elucidation of the roles of the two ARID3B isoforms will help us understand ovarian cancer progression, cellular response to chemotherapy, and develop prognostic markers and targeted therapies.

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 2183. doi:1538-7445.AM2012-2183
Abstract 2184: Analysis of human polynucleotide phosphorylase function in human melanoma
Authors: Sokhi Upneet K.
Published: August 2012

Human polynucleotide phosphorylase (hPNPaseold-35) is an evolutionarily conserved 3′, 5′ exoribonuclease predominantly involved in mRNA degradation. It is a type I IFN-inducible early response gene that was identified as a gene upregulated in terminally differentiated and senescent fibroblasts. Overexpression of hPNPaseold-35 via a replication incompetent adenovirus (Ad.hPNPaseold-35) arrests multiple cancer cell types in the G1 phase of the cell cycle, with cells eventually undergoing apoptosis. A potential mechanism of this growth inhibition and eventual apoptosis induction is the ability of hPNPaseold-35 to selectively degrade c-myc mRNA. This was further proven by the overexpression of c-myc that partially but significantly protects human melanoma cells from Ad.hPNPaseold-35-mediated growth inhibition, indicating that c-myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Since the protection offered by c-myc overexpression was incomplete, additional targets of hPNPaseold-35 are likely to exist that may play a role in mediating its growth-inhibitory effects. The goal of the present study was to identify these additional candidate genes that may be directly or indirectly regulated by hPNPaseold-35, which would help us better understand the physiological implications of this interesting enzymatic protein. In order to identify the target genes a whole genome cDNA microarray was performed to assess gene expression changes that occur when hPNPaseold-35 was silenced in HO-1 melanoma cells. The genes that are upregulated in the hPNPaseold-35 knockdown cell line might be potential direct degradation targets of hPNPaseold-35 according to our current hypothesis. Finally, validation of these targets may provide us a unique opportunity for defining specific molecules and pathways that may represent new targets of hPNPaseold-35 contributing to its growth-inhibitory effects, which may be further used for developing approaches to enhance its therapeutic efficacy.

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 2184. doi:1538-7445.AM2012-2184
Abstract 2188: The ubiquitin-specific protease USP2a targets cyclin A1: A potential role in bladder cancer
Authors: Kim Jayoung , Loda Massimo F. , Freeman Michael R.
Published: August 2012

A deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX, or Aurora-A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about USP2a's role or mechanism in bladder cancer. Here we provide evidence that USP2a functions as an oncoprotein in bladder cancer cells. Overexpression of USP2a caused enhanced proliferation, invasion, migration, and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity, and reduced migratory/invasive capability, compared to control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by blocking its degradation. Enforced expression of wild type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. Our finding that USP2a impairs ubiquitination and stabilizes the important cell cycle regulator, cyclin A1, raises the possibility of USP2a silencing as a therapeutic strategy against bladder tumors in combination with chemotherapy.

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 2188. doi:1538-7445.AM2012-2188
Abstract 2192: Genomic amplification and drug-resistance roles of the KDM5A histone demethylase gene in breast cancer
Authors: Hou Jinling , Wu Jack , Dombkowski Alan , Boerner Julie L. , Ethier Stephen , Yang Zeng-Quan
Published: August 2012

Interconnected genetic and epigenetic events control both the initiation and progression of cancer. Specifically, genetic alterations, such as amplification, and the subsequent over-expression of genes encoding ‘epigenetic modifying enzymes’ can directly lead to histone code changes that may be critical for cancer progression. KDM5A (lysine (K)-specific demethylase 5A, also known as RBP2 and JARID1A) was originally identified as the retinoblastoma-binding protein (RBP) implicated in the regulation of retinoblastoma target genes. Recently, several groups discovered that KDM5A is a member of a set of newly identified histone demethylases that control the chromatin-mediated regulation of gene expression. Specifically, KDM5A can function as a transcriptional repressor through the demethylation of tri- and dimethylated histone H3 at lysine 4 (H3K4) active marks. In this study, we observed a significant amplification and over-expression of the KDM5A gene in various tumors, including breast cancer. We found that breast cancer cells with KDM5A gene amplification had intrinsic drug resistance properties and knocking down KDM5A with shRNAs improved the efficacy of epidermal growth factor receptor (EGFR) inhibitors against these breast cancer cells. Further, up-regulation of KDM5A modified the histone methylation status and altered the expression of a subset of key genes, including the tumor suppressor p21 and the apoptotic effector BAK1, in breast cancer. Our findings suggest that alteration of the KDM5A gene may have a critical role in the pathogenesis of 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 2192. doi:1538-7445.AM2012-2192
Abstract 2195: Smurf2 is a novel tumor suppressor gene that governs chromatin structure and genome integrity through RNF20
Authors: Blank Michael , Zhang Ying E.
Published: August 2012

Cancers are known to harbour alterations in chromatin landscape in addition to allelic mutations. Here, we show that genomic ablation of Smurf2, a HECT-domain E3 ubiquitin ligase, results in dysregulation of global gene expression, altered DNA damage response, and unstable genomes, which culminate to increased susceptibility to various types of cancers in aged mice. We demonstrate that Smurf2 regulates histone H2B monoubiquitination as well as histone H3 tri-methylation at K4 and K79 by targeting RNF20, the major E3 ligase for histone H2B ubiquitination, to proteasomal degradation. We further show that Smurf2 and RNF20 are co-localized at the γ-H2AX foci of double-stranded DNA breaks in the nucleus, and Smurf2 regulates RNF20 stability in human cancer cells. Thus, our data indicate that Smurf2 has a tumor suppression function that normally maintains genomic stability by controlling the epigenetic landscape of histone modifications through RNF20.

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 2195. doi:1538-7445.AM2012-2195
Abstract 2197: Fliz-1 regulation of GATA-3 expression in mammary tumor cells
Authors: Shrestha Eric , Wang TianJiao , Saunders Justin , Knepper Janice E.
Published: August 2012

The GATA-3 transcription factor is a master regulator of cell fate determination in the mammary gland. Mice lacking expression of GATA-3 in the mammary gland fail to develop complete ductal branching or terminal end buds, while depletion of GATA-3 from adult mammary glands leads to loss of markers for luminal cell differentiation and to caspase-mediated death of luminal cells. In human breast cancer, GATA-3 expression is found in highly differentiated, estrogen receptor positive tumors with good prognosis, while loss correlates with highly metastatic tumors resistant to endocrine therapy. Regulation of GATA-3 expression is incompletely understood, though a positive-cross regulatory loop with the estrogen receptor alpha has been defined. The zinc finger protein Fliz-1 represses GATA-3 transcription in the T cell lineage, but its role in mammary cells is unknown. We hypothesize that Fliz-1 also can repress GATA-3 expression in mammary cells, leading to loss of estrogen response. To test this hypothesis, we identified a mouse mammary tumor cell line expressing high levels of Fliz1, and transfected the cells with a Fliz-1 silencing vector or negative control. Successful knockdown was confirmed by RT/PCR. Cells with decreased Fliz-1 expression showed increased expression of GATA-3 and estrogen receptor alpha mRNA relative to controls. The Fliz-1-expressing cells formed tumors that were insensitive to tamoxifen treatment when implanted in syngeneic BALB/c mice, while cells with decreased Fliz-1 expression exhibited decreased tumor growth in response to endocrine therapy. As a reciprocal experiment, we constructed a fusion gene linking Fliz-1 with a C-terminal green fluorescent protein tag. The fusion protein properly localizes to the nucleus of transfected MCF-7 (human breast cancer) and COMMA-D (mouse mammary epithelial) cells. Chromatin immunoprecipitation was used to show that Fliz1-GFP binds to an intronic regulatory region within the GATA-3 gene in COMMA-D cells. These data provide evidence supporting a role for Fliz-1 in control of GATA-3 expression in mammary cells and tumors.

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 2197. doi:1538-7445.AM2012-2197
Abstract 2198: A functional ETS/E2F module in cancers expressing ETS fusion genes
Authors: Schwentner Raphaela , Bilke Sven , Kauer Max , Jug Gunhild , Walker Robert L. , Meltzer Paul S. , Kovar Heinrich
Published: August 2012

Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly binds to 50% of E2F target genes in ESFT. Furthermore, several E2F factors were themselves found to be directly regulated by EWS-FLI1. Therefore, we studied the functional interaction of EWS-FLI1 with E2F. Ten arbitrarily chosen EWS-FLI1/E2F candidate targets including E2F3, RAD51, and ATAD2 were chosen for an in-depth promoter activity study by luciferase reporter gene assays in combination with mutation analysis of transcription factor binding motifs. By ChIP-PCR we confirmed direct EWS-FLI1 promoter binding, and observed decreased reporter activity for all ten studied promoters upon knockdown of EWS-FLI1 by RNAi. Furthermore, the study of promoter occupancy by different E2F family members revealed that silencing of EWS-FLI1 results in the exchange of EWS-FLI1 induced, activating E2F3 by constitutively expressed repressive E2F4 on the promoters of there jointly regulated target genes. Importantly, testing E2F3 promoter occupancy on wildtype and ETS motif mutated promoter constructs in ESFT cell lines revealed that binding of E2F3 to its target promoters is dependent on an intact ETS binding site. Mutation of the ETS motif resulted in reduced binding of E2F3. These data are consistent with a model in which EWS-FLI1 actively recruits an activating E2F factor thereby replacing a repressing E2F factor and in which EWS-FLI1 binding is essential for E2F binding. Strikingly, the functional E2F/ETS transcriptional module detected in ESFT for the chimeric ETS factor EWS-FLI1 was found to be also relevant to TMPRSS2-ERG expressing prostate cancer cells, but not active in HeLa cells. Supported by grants from the Austrian Science Fund (P22328-B09), and the European Comission (EU-FP7 STREP 259348). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences.

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 2198. doi:1538-7445.AM2012-2198
Abstract 2202: HMG box-containing protein 1 (HBP1) is a direct target of transcription factor FOXO1 in invasive oral cancer
Authors: Chou I-Tai , Chan Chien-Yi , Lee Ming-Fen , Huang Chun-Yin
Published: August 2012

Constitutively activated PI3K/Akt signaling with deregulated activity of FOXO factors is often associated with tumorigenesis. Reduced HBP1 expression and/or loss-of-function HBP1 mutants have been observed in several types of cancer. FOXO1 and HBP1 seem to share common biological roles such as cell growth inhibition. We have previously demonstrated that, in oral cancer, reduced HBP1 expression expedited cell proliferation, growth, and migration, while the role of FOXO1 remains unclear. Hence, the objective of the current study is to investigate the reciprocal roles of FOXO1 and HBP1 in oral cancer. In a subset of oral tumor specimen, we found that both FOXO1 and HBP1 expression levels were significantly lower than those of the control tissues. In addition, FOXO1 expression was positively correlated with that of HBP1, and the status of low FOXO1 and HBP1 was associated with invasiveness of oral tumor, indicating a coordinated down-regulation of FOXO1 and HBP1 in invasive oral cancer. To further examine if HBP1 is a transcriptional target of FOXO1, we searched for putative binding sites within a 2-kb HBP1 promoter region, and identified two FOXO1 consensus sites, located −177 to −184 bp and −388 to −395 bp, from the transcription start site. As shown by reporter analysis and in vivo chromatin IP assay, these cis-acting elements and trans-acting FOXO1 factors, with functional DNA-binding domains, were necessary for complete activation. Furthermore, suppression of FOXO1 led to a reduction of HBP1 expression. In a reciprocal manner, FOXO1-mediated inhibition of colony formation, cell growth, and invasion was disrupted upon HBP1 knockdown in invasive oral cancer cells. Taken together, our data provide the significant findings that HBP1 is a novel downstream target of FOXO1 and that the FOXO1-HBP1 pathway might be a prospective means of intervention in invasive oral 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 2202. doi:1538-7445.AM2012-2202
Abstract 2206: Molecular interaction of HIF-1α and the androgen receptor in prostate cancer
Authors: Price Douglas K. , Reece Kelie M. , Troutman Sarah M. , Pressler Heather M. , Pisle Stephen T. , Figg William D.
Published: August 2012

Background: Prostate cancer is the most common noncutaneous cancer among men in the United States, and its progression is largely controlled by the androgen receptor (AR). Androgen deprivation therapy (ADT) is an initially effective treatment for prostate cancer, but most tumors eventually become castrate resistant. Tumor hypoxia also appears to be associated with a poor prognosis in prostate cancer. HIF-1α regulates the transcription of genes that allow tumor survival and growth in low oxygen conditions. Our laboratory has data showing that in response to castration and anti-androgen therapy in mice, there was a strong transcriptional relationship between HIF-1α and AR, as measured by quantitative RT-PCR, suggesting an interaction between the two proteins. Thus, the purpose of this study was to determine if there is a molecular interaction between HIF-1α and AR in prostate cancer cells. Methods: We used Western blot analysis to examine the expression levels of HIF-1α and AR in LNCaP prostate cancer cells to determine if they are upregulated together at the protein level. Four experimental conditions were tested: control (no treatment), DHT for induction of AR expression, CoCl2 for induction of HIF-1α, and a combination treatment of DHT and CoCl2. Immunoprecipitation experiments were carried out to determine if there is an association between HIF-1α and AR. In addition, cells were fractionated into nuclear and cellular cells extracts, followed by Western blot analysis to determine where in the cells this interaction occurs. Results: Western blot analysis of cell lysates showed synergistic upregulation of both HIF-1α and AR expression only under combined CoCl2 and DHT treatment conditions. In addition, immunoprecipitation experiments showed that HIF-1α and AR exist in a complex with one another, and fractionation experiments indicated this complex occurs in the nucleus. Conclusions: Our results demonstrate that HIF-1α and AR associate with one another in cells. Binding assays are in progress to determine the nature of this interaction. In addition, we are examining the cellular consequences of this protein interaction, as it is possible that upregulation of HIF-1α in response to low androgen contributes to the development of tumor resistance to ADT.

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 2206. doi:1538-7445.AM2012-2206
Abstract 2208: JNK2 regulates the hif-1α mRNA stability in a nucleolin-dependent manner
Authors: Zhang Dongyun , Li Jingxia , Zhang Min , Zuo Zhenghong , Yu Yonghui , Huang Chuanshu
Published: August 2012

HIF-1α mRNA is regarded as constitutively and ubiquitously expressed no matter the level of oxygen tension. But most recently, more and more labs found that HIF-1α mRNA could be regulated by natural antisense transcripts, potential microRNAs, and low O2 pressure in the renal medulla in rats and in sea bass. In our current studies, we found that nucleolin, the classic histone chaperon, was able to physically bind to hif-1α mRNA and maintained its stability. Knocking down nucleolin by shRNA obviously reduced HIF-1α protein induction in response to nickel treatment resulting from the impaired expression of hif-1α mRNA. Both the promoter luciferase assay and mRNA degradation rate assay by ActD clearly showed that nucleolin knocking down did not change hif-1α transcription, but rather affected its mRNA stability. Further work found JNK2 could regulate nucleolin expression and subsequently affect its function in stabilizing hif-1α mRNA, by which we provided more evidence for the oncogenic role of JNK2 and nucleolin in regulating the cancer environments by controlling HIF-1α expression.

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 2208. doi:1538-7445.AM2012-2208
Abstract 2209: Cellular characterization of chromatin state via high throughput ChIP assay
Authors: Li Zirong , Rosenfeld John , Kovelman Robert
Published: August 2012

Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated. Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously. Recently, we have developed a simple high throughput chromatin immunoprecipitation protocol that utilizes high capacity protein A/G coated magnetic beads and 96 well plates. This protocol requires fewer steps and less hands-on time, has higher sensitivity and more reliable performance as compared to conventional approaches. The method is also compatible with multi-channel pipetting and liquid handling systems. We have successfully used this protocol to map various clinically relevant chromatin marks and controls across several cell types to quantitatively measure chromatin states. This analysis included a variety of marks corresponding to repressed, poised and active promoters, strong and weak enhancers, putative insulators, transcribed regions, as well as large-scale repressed and inactive domains. This study demonstrates the utility of this approach for the characterization of model cellular systems in perturbation studies with chemical probes.

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 2209. doi:1538-7445.AM2012-2209
Abstract 221: p21-activated kinase 1 (Pak1) phosphorylates BAD directly at serine 111 in vitro and indirectly through Raf-1 at serine 112
Authors: Field Jeffrey M. , Ye Diana Z. , Jin Shenghao , Zhou Ya
Published: August 2012

Cell survival depends on the balance between protective and apoptotic signals. When the balance of signals tips towards apoptosis, cells undergo programmed cell death. This balance has profound implications in diseases including cancer. Oncogenes and tumor suppressors are mutated to promote cell survival during tumor development, and many chemotherapeutic drugs kill tumor cells by stimulating apoptosis. BAD is a pro-apoptotic member of the Bcl-2 family of proteins, which can be phosphorylated on numerous sites to modulate binding to Bcl-2 and 14-3-3 proteins and inhibit its pro-apoptotic activities. One of the critical phosphorylation sites is the serine 112 (S112), which can be phosphorylated by several kinases including Pak1. We mapped the Pak phosphorylation sites by making serine to alanine mutations in BAD and testing them as substrates in in vitro kinase assays. We found that the primary phosphorylation site is not S112 but serine 111 (S111), a site that is sometimes found phosphorylated in vivo. In transfection assays of HEK293T cells, we showed that Pak1 required Raf-1 to stimulate phosphorylation on S112. Mutating either S111 or S112 to alanine enhanced binding to Bcl-2, but the double mutant S111/112A bound better to Bcl-2. Moreover, BAD phosphorylation at S111 was observed in several other cell lines, and treating one of them with the Pak1 inhibitor 2,2α-Dihydroxy-1,1α-dinaphthyldisulfide (IPA-3) reduced phosphorylation primarily at S112 and to a smaller extent at S111, while Raf inhibitors only reduced phosphorylation at S112. Together, these findings demonstrate that Pak1 phosphorylates BAD directly at S111, but phosphorylated S112 through Raf-1. These two sites of BAD serve as redundant regulatory sites for Bcl-2 binding.

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 221. doi:1538-7445.AM2012-221
Abstract 2210: Global levels of H3K27me3 track with differentiation in vivo and are deregulated by MYC in prostate cancer
Authors: Pellakuru Laxmi Gurunatham , Iwata Tsuyoshi , Gurel Bora , De Marzo Angelo M.
Published: August 2012

Both embryonic stem cells and various cancer cell types contain abundant amounts of decondensed chromatin. However, the molecular mechanisms responsible for altering chromatin states in stem cells and cancer cells are not well understood. One factor may be the oncogenic transcription factor, MYC. MYC is overexpressed in many tumor types and affects a wide array of essential cellular processes including ribosome and mitochondrial biogenesis, metabolism, and cell cycle progression. MYC is also a key factor in reprogramming of somatic adult cells into induced pluripotent stem cells. Less well known is that MYC also affects global chromatin structure where it is required to maintain open or active chromatin. We hypothesize that in prostate cancer, MYC affects global chromatin structure keeping it in a more open conformation thus maintaining the cells in a less differentiated state resembling that of stem cells. We show by immunohistochemistry that compared to the more differentiated luminal cells, less differentiated stem-like cells in the basal compartment of the human and mouse prostate contain lower levels of the polycomb heterochromatin mark, H3K27me3. This link to the differentiated state of normal cells is also found in a number of other human and rodent tissues characterized by hierarchical differentiation and rapid turnover of stem cell compartments. Further, in MYC-driven prostate cancers in mice, global levels of H3K27me3 are reduced in both prostatic intraepithelial neoplasia (PIN) and invasive adenocarcinoma lesions. This pattern of global H3K27me3 reduction in PIN and invasive prostate cancers is also observed in humans, correlates with global chromatin decondensation, and correlates with increased markers of disease aggressiveness (e.g. Gleason score and pathological stage). In prostate and breast cancer cells, experimentally forced reductions in MYC levels result in increased global levels of H3K27me3. These findings suggest that increased levels of decondensed chromatin in normal stem/progenitor cells and in cancer cells are associated with global loss of the H3K27me3 polycomb mark, and furthermore, MYC overexpression could be one mechanism for this H3K27me3 deregulation.

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 2210. doi:1538-7445.AM2012-2210
Abstract 2217: Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer
Authors: Patrick Brad A. , Jaiswal Anil K.
Published: August 2012

In previous studies we have illustrated a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in stabilization of tumor suppressor transcription factor p63 against 20S proteasomal degradation, resulting in thinning of epithelium and chemical-induced skin cancer [Oncogene (2011) 30, 1098-1107]. Current studies demonstrate that an intermediate pathway involving NQO1 control of C/EBPα against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and profound decrease of p63 gene expression. p63 promoter mutagenesis, transfection and ChIP assays identified C/EBPα binding site between nucleotide position −185 to −174 that allows C/EBPα to enhance p63 gene expression. Coimmunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPβ. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against ubiquitin-independent 20S proteasomal degradation and NQO1 protection of C/EBPα required binding of cofactor NADH. Exposure of skin and keratinocytes to stress agents benzo(a)pyrene, UVB, antioxidant t-BHQ and ionizing radiation led to induction of NQO1 and stabilization of C/EBPα protein resulting in an increase in p63 RNA and protein in wild type but not in NQO1-null mice. Collectively, the current data combined with previous suggest that stress-mediated induction of NQO1 acts both through stabilization of p63-transactivating C/EBPα and through direct stabilization of p63 to control keratinocyte differentiation leading to normal skin development and possibly contribute to protection against chemical/radiation-induced skin carcinogenesis. The studies are significant in light of the knowledge that 2-4% human individuals are homozygous and 23% are heterozygous for NQO1P187S mutation and might be susceptible to stress-induced skin diseases.

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 2217. doi:1538-7445.AM2012-2217