bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3122: Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system
Authors: Monsey John , Shen Wei , Schlesinger Paul S. , Bose Ron
Published: February 2011

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor tyrosine kinases belonging to the Epidermal Growth Factor Receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains have demonstrated kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. In vitro studies of EGFR tyrosine kinase dimerization and activation were previously reported using a nickel-chelating lipid - liposome system, and we now demonstrate this system can be broadly applied to all other members of the EGFR family. Polyhistidine tagged-Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-chelating lipid containing liposomes and are brought to a high local concentration, mimicking what happens to the full-length receptors in vivo following ligand binding. Addition of Nickel-liposomes to Her4 kinase domain results in a 40-fold activation in kinase specific activity and a marked enhancement in C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process that requires kinase dimerization. Her2/neu kinase activity is also activated by Nickel-liposomes, and further increases in Her2/neu activity are achieved by heterodimerization with either Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model, and suggests that the mode of dimerization of Her3 needs to be further studied.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3122.
Abstract 3123: Insulin-like Growth Factor-I Suppresses Bone Morphogenetic Protein Signaling through a Phosphatidylinositol 3-Kinase, Akt and mTOR-Dependant Mechanism
Authors: Wahdan-Alaswad Reema S. , Song Kyung , Krebs Tracy L. , Danielpour David
Published: February 2011

Bone morphogenetic proteins (BMPs) and insulin-like growth factor-I (IGF-I) function as critical regulators of prostate tumor cell growth. BMPs have recently been shown to be pivotal in controlling prostate tumorigenesis, and loss of BMP receptor function has been correlated to a higher Gleason grade in prostate cancer patients. The mechanism of such loss of BMP function in prostate cancer is unclear, and is likely to occur through multiple mechanisms. One likely candidate we are investigating is the tumor suppressor PTEN, which is lost up to 50% of patients with advanced prostate cancer. Loss of PTEN leads to enhanced activation of PI3K, Akt signaling and suppression of apoptosis. We provide the first evidence that IGF-I intercepts BMP4-induced apoptosis in prostate epithelial cell lines, as shown by changes in cell number, Hoechst 33342 staining, cell viability and flow cytometry. We show that IGF-I suppresses BMP4-induced activation of Smads 1, 5 and/or 8, and that such suppression is reversed by chemical inhibitors of PI3K, Akt or mTOR, or by wild-type PTEN or a dominant-negative PI3K expression construct efficiently delivered to cells by transduction adenoviral. We further show that IGF-I represses BMP4-induced transcriptional activation of the BMP targets, Id-1, Id-2, and Id-3. These results suggest that IGF-I signaling via a PI3K, Akt and mTOR-dependent mechanism inhibits transcriptional activity and apoptosis by BMP4 through suppressing the activation of Smads 1, 5 and/or 8 in prostate epithelial cells. All in all, our findings have important implications in the therapeutic intervention of prostate cancer by restoring BMP-mediated tumor suppressive capabilities by blocking IGF-I signaling pathway's oncogenic activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3123.
Abstract 3129: Regulation of expression and role of the pigment epithelium derived factor in Ras-mediated pancreatic oncogenesis
Authors: Maya Joanna , Robin Tyler , Crawford Susan , Campbell Paul M. , Der Channing J. , Grippo Paul , Repasky Gretchen A.
Published: February 2011

Mutated and constitutively activated Ras oncoproteins are found in ∼33% of all human cancers. In particular, Ras mutations are found in a majority of pancreatic cancers (90%), and aberrant Ras activation has been shown to be critical for promoting pancreatic adenocarcinomas. Ras-mediated transformation is associated with downregulation of Pigment Epithelium Derived Factor (PEDF), an anti-angiogenic and lipid regulatory factor shown to suppress oncogenic K-Ras-induced invasive disease in vivo. PEDF has also been shown to have anti-tumorigenic activity on tumor cells themselves. Specifically, mice with chronically active mutant KRAS(12D) background form precancerous lesions with high frequency, and knockout of PEDF expression specifically in pancreatic tissue resulted in a significant aggravation of the precancerous phenotype. The mechanism(s) of this biological phenotype is unclear and the specific role of PEDF as a potential tumor suppressor gene in Ras-mediated oncogenesis remains unresolved. Western analyses were used in this study to investigate the expression of PEDF in K-Ras-transformed and nontransformed human pancreatic duct-derived cells (HPNE) and their surrounding cell culture growth media. We observe that PEDF is upregulated both intracellularly under growth and serum-starved conditions and extracellularly under serum-starved conditions in K-Ras-transformed human pancreatic cells. We are also investigating the role of exogenous PEDF in cell proliferation induced by oncogenic variants of K-Ras. While no significant difference was observed in cell proliferation between the PEDF-treated and nontreated cells (whether they be K-Ras-transformed and nontransformed cells) in growth conditions, a decrease in cell proliferation was observed in the PEDF-treated K-Ras-transformed cells under serum-starved conditions. By better understanding the link between Ras and PEDF we will be able to assess the contribution and therapeutic potential of PEDF to formation and progression of pancreatic cancer while investigating novel targets for therapeutic intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3129.
Abstract 3141: Hyperglycemia modulates the Wnt /b-catenin signaling pathway in breast cancer
Authors: Vaira Sergio , Friday R. Ellen , Scott Keith , Conrad Steven , Mills Glenn , Turturro Francesco
Published: February 2011

Hyperglycemia represents one of the early events in the development of diabetes type 2. Susceptibility to this disease has been linked to Wnt/β-catenin signaling pathway that also controls breast cancer progression. We had previously analyzed the effects of hyperglycemia on gene expression profile (GEP) by Affimetrix analysis in breast cancer derived MDA-MB-231 cells (Turturro F. et al., BMC Cancer 2007, 7:96). Wnt/βcatenin signaling pathway had emerged as one of the most prominent signaling pathway regulated by increased glucose in culture media (5 mM versus 20 mM).

The aim of the present study was to validate the GEP data by assessing the mRNA levels of both GSK-3b and Wnt1 by RT-PCR in response to hyperglycemia as previously described. We analyzed the levels of RT-PCR products of the target genes relative to the control mRNA of β-actin in MDA-MB-231 cells, cultured in the same conditions as previously illustrated (Turturro F. et al., BMC Cancer 2007, 7:96). The values were expressed as densitometric readings. Relative levels of GSK-3b mRNA raised from 0.14 + 0.053 for 5 mM glucose to 0.308 + 0.0476 for 5/20 shift (p< 0.05 at 5 vs. 5/20 mM) and to 0.432 + 0.0321 for 20 mM (p< 0.05 at 5 vs. 20 mM) resulting in an overall 3 fold increase. We also examined the effect of hyperglycemia on gene expression of Wnt-1 which inhibits the expression and activity of GSK-3b. The relative levels of mRNA of Wnt-1 assessed by RT-PCR, were decreased from 0.163 + 0.0322 for 5 mM glucose to 0.068 + 0.001 for 20 mM glucose, with an overall 2.7 fold reduction (p<.05 at 5 vs. 20 mM).

These data confirmed the GEP results in terms of relationship between hyperglycemia and gene expression of the Wnt signaling pathway in this particular breast cancer cell line. Our study supports further investigation in the area of diabetes and breast cancer invasiveness and progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3141.
Abstract 3142: Abnormal expression of the ALK protein in ovarian carcinoma is associated with low copy number amplification of the 2p23 locus
Authors: Merkel Patricia , Grubinska Barbara , Drew Allison , Szilvassy Stephen J. , Gyuris Tibor , Merriam Kim , Tsui Kim , Rottman James , Parker Alex , Brake Rachael
Published: February 2011

Anaplastic lymphoma kinase (ALK) is predominantly expressed in the brain and spinal cord during neonatal development and is subsequently down-regulated in the adult. In cancer, ALK is ectopically expressed as a consequence of one of several different cytogenetic translocations or inversions that lead to the expression of an oligomerized, constitutively active, intracellular tyrosine kinase. The resulting fusion protein has been identified in anaplastic large cell lymphomas, non small cell lung carcinomas and more recently breast and colorectal carcinomas. Preclinical data convincingly demonstrates that fusion transcripts can drive tumorigenesis and inhibition of ALK in vitro and in vivo leads to inhibition of cellular growth. Full length (fl) ALK is also expressed in a variety of other tumor types, and recent data suggest that at least in neuroblastoma, the flALK transcript contains activating point mutations in the tyrosine kinase domain. These data suggest that flALK may also be a therapeutic target in cancer. Here we demonstrate that a significant subset of ovarian carcinoma cell lines and biopsy specimens aberrantly express ALK. Gene expression analysis in 126 ovarian carcinoma samples demonstrated a prevalence of ALK expression of 19%. Analysis of the histological subtypes within this study, showed specific enrichment of ALK transcript in papillary ovarian carcinoma (15 of 42 samples). This subtype showed a range of ALK expression between 2 - 4 fold increase above normal ovarian tissue. We have also confirmed that in a subset of ovarian tumor cell lines and primary tumor samples that ALK transcript is converted into ALK protein. DNA sequence analysis has revealed that in these patient samples and cell lines, ALK is predominantly wild type. Single nucleotide polymorphism (SNP) analysis and fluorescent in situ hybridization (FISH) suggest that over expression may occur as a consequence of low copy number amplification. These preliminary findings suggest that ALK expression in cancer has now been observed in ovarian carcinomas by a mechanism distinct from cytogenetic translocations or inversions. Currently, we are attempting to correlate the aberrant expression of ALK with ovarian cancer prognosis as well as understand the value of inhibiting ALK expression in this cell type.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3142.
Abstract 3145: Non-coding promoter-associated RNAs (PARs) orchestrate the epigenetic control of CDH1 transcription in cancer cells
Authors: Magistri Marco , Pastori Chiara , Napoli Sara , Carbone Giuseppina M. , Catapano Carlo V.
Published: February 2011

DNA in eukaryotic cells is wrapped around histones to form chromatin fibers. Chromatin can assume different conformations that dictate its biological activity. The equilibrium between distinct chromatin states is achieved via a cascade of epigenetic events involving DNA and histone modifications. However, the underlying mechanisms determining the timing and selectivity of these epigenetic events are not fully understood. Alterations in epigenetic patterns are common in cancer leading to the aberrant silencing of tumor suppressor genes. The CDH1 gene encodes the epithelial cell adhesion molecule E-cadherin and is frequently silenced in epithelial tumors. CDH1 silencing is associated with cell transformation, tumor invasiveness and metastatic dissemination along with epithelial-mesenchymal transition and acquisition of stem cell properties. Recently, non-protein coding RNAs (ncRNAs) and components of the RNA interference machinery have been proposed as important elements in epigenetic mechanisms that might assist in targeting epigenetic effectors to specific genomic loci. In this study, we investigated the role of non-coding promoter-associated RNA (PARs) in the transcriptional regulation of the CDH1 gene. PARs with both sense and antisense orientation relative to the protein-coding RNA were found in the CDH1 gene using strand-specific RT-PCR and 5’RACE. Sense and antisense PARs were positively and negatively correlated, respectively, to the level of CDH1 RNA in different cell lines, suggesting a functional link between coding and non-coding RNAs. RNA immunoprecipitation showed binding of the Argonaute protein Ago1 to the sense PAR in PC3 cells, in which the CDH1 gene was silenced. Knock-down of Ago1 led to re-expression of CDH1 along with increased histone H3 acetylation and decreased histone H3K9 methylation in the CDH1 promoter. Similar changes in the histone code and re-expression of CDH1 were induced by knocking down the H3K9 histone methyltransferase SUV39H1 and HDAC1. Moreover, Ago1 knock-down reduced binding of both SUV39H1 and HDAC1 to the CDH1 promoter, indicating a critical role of Ago1 in establishing their association with the promoter. Consistent with a role of the PARs in CDH1 transcriptional control, targeting the sense PAR with small interfering RNAs induced chromatin remodeling and CDH1 re-expression, supporting the hypothesis that PARs can direct epigenetic effectors to gene promoters through their interaction with Ago1. These findings suggest the existence of endogenous ncRNA-based mechanisms of transcriptional control that cooperate with and complement DNA and protein based epigenetic pathways in human cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3145.
Abstract 3146: The role of SUMO on the stabilization of estrogen receptor β cx in hypoxic conditions
Authors: Dey Prasenjit , Strom Anders M. , Gustafsson Jan-Ake
Published: February 2011

Estrogen receptor (ER) βcx is a 495 amino acid splice variant of estrogen receptor (ER) β. It is highly expressed in testes and hypoxic brain, with small amount of expression observed in prostate, thymus and ovary. Clinical studies in breast cancer, shows that, increased expression/stabilization and cytosolic localization of ERβcx have a bad prognosis compared with nuclear localization. We believe that hypoxia play a role in the stabilization of ERβcx. As hypoxia is central to cancer tumorigenesis, the role played by ERβcx in hypoxic environment can elucidate, at least partially, cancer progression and prognosis. We found that, HEK293T cells transiently transfected with YFP-ERβcx fusion protein show more intense fluorescence in the presence of 100 μM CoCl2 (a hypoxia mimetic) indicating the stabilization of the YFP-ERβcx protein. As a control the YFP-ERβ do not show any change after treatment with CoCl2. The ERβcx differ from ERβ by an alternatively spliced last exon. It is well known from previous studies that hypoxic condition stabilizes HIF1α, a transcriptional regulator of a cell's adaptive response to hypoxia. This may causes upregulation of small ubiquitin-related modifier (SUMO) pathway. A close examination of the unique CX exon reveals a SUMOylation consensus sequence. Mutation of this sequence abolishes the hypoxic regulation of ERβcx. Elucidating the regulatory mechanism of ERβcx might give clues to its role in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3146.
Abstract 3150: The SCFFbw7 E3 Ubiquitin Ligase Complex Targets KLF5 for Ubiquitination and Degradation in a Phosphorylation Dependent Manner
Authors: Zheng Han-Qiu , Zhao Dong , Zhou Zhongmei , Chen Ceshi
Published: February 2011

The KLF5 transcription factor is overexpressed in ERα negative basal type breast cancer and promotes breast cell proliferation, survival, and tumorigenesis through upregulating its target gene FGF-BP. We previously identified WWP1 as an E3 ubiquitin ligase for KLF5. However, the regulation mechanisms of KLF5 protein degradation through the ubiquitin proteasome pathway are still poorly understood. In this study, we demonstrate that the Skp1-Cul1-Fbw7 E3 ubiquitin ligase complex (SCFFbw7) targets KLF5 for ubiquitin-mediated degradation in a KLF5 phosphorylation dependent manner. Fbw7 interacts with KLF5 through the WD40 repeats of Fbw7 and a cdc-4 phospho-degron(CPD domain, 303SPPSS) of KLF5 and promotes KLF5 ubiquitination and degradation. Both the F-box and WD40 repeats of Fbw7 are required for KLF5 ubiquitination and degradation. Mutation of the critical Ser303 residue in the KLF5 CPD motif into Ala abolishes the protein interaction, ubiquitination, and degradation by Fbw7. In addition, the interaction between wild type KLF5 and Fbw7 can be abrogated by alkaline phosphotase (CIP) treatment. Using a KLF5 S303 phosphorylation specific antibody, we demonstrate that the S303 residue is phosphorylated. We further demonstrate that the KLF5 S303 can be phosphorylated by GSK3 in vitro. Inhibition of GSK3 activity by LiCl delays the KLF5 S303 phosphorylation and the KLF5 protein degradation. This new regulatory mechanism of KLF5 degradation may result in useful diagnostic and therapeutic targets for breast cancer and other cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3150.
Abstract 3153: The role of ubiquitin in androgen receptor function in prostate cancer
Authors: Darby Steven , Lumsden Hollie , Gaughan Luke , Robson Craig N.
Published: February 2011

IntroductionProstate cancer (PC) is the most common male cancer in the western world. The androgen receptor (AR) plays a central role in PC development and remains the primary target for therapy. Androgen deprivation therapy is currently the standard treatment for PC. However, following a 2-3 year period, a more aggressive cancer usually recurs that is unresponsive to further androgen deprivation and is termed castrate resistant prostate cancer (CRPC). Several AR interacting proteins are components of the ubiquitin-proteasome degradation system, including Mdm2, CHIP and E6AP E3 ligases implicating proteasomal degradation/activation as a key event in AR function. In this study we have investigated which ubiquitin ligases are involved in AR ubiquitination and whether altered ubiquitination levels play a critical role and subsequent AR function in development of CRPC.MethodsIn vitro ubiquitination assays were performed with AR as the substrate with multiple purified E3 ligase enzymes in conjunction with wild type or mutant ubiquitin (K7R, K48 and K63). A panel of E2 conjugating enzymes were also employed to identify which E2/E3 heterodimers were involved in this process. Ubiquitinated AR fragments were gel excised and digested with trypsin. Fragments were then subjected to proteomic analysis to determine the sites of ubiquitination in AR.ResultsIn vitro ubiquitination assays using lysine null ubiquitin (K7R) revealed monoubiquitination of AR in multiple domains of AR in response to four E3 ligases. Screening of an E2 panel also identified multiple E2 conjugating enzymes implicated in AR ubiquitination. K48 ubiquitin (implicated in proteosomal degradation) and K63 ubiquitin (implicated in substrate activation) incorporated into in vitro assays identified differential poly-ubiquitination chain patterns in AR, dependent on which E3 ligase was used in the assay. Proteomic analysis of ubiquitinated AR fragments identified a novel site of ubiquitination within AR.ConclusionsAR was ubiquitinated by four E3 ligases evaluated in this study. Mono- and poly-ubiquitination of AR was identified. Mass spectroscopy revealed a novel site of ubiquitination in AR. Further experiments are being performed, including site directed mutagenesis to determine the importance of the novel ubiquitination site in AR protein proteosomal turnover and transcriptional activation.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3153.
Abstract 3154: Translation of TRAF1 is regulated by IRES-dependent mechanism and stimulated by vincristine in lymphoid malignancies
Authors: Yang Lin , Gu Lubing , Li Zhouya , Zhou Muxiang
Published: February 2011

TRAF1 is a member of the TRAF family, which plays important roles in signal transduction that mediate cell life and death in the immune response, inflammatory and malignant diseases. It is known that TRAF1 transcription is inducible by various cytokines, but little is known about the regulation of its protein translation. In the present study, we demonstrated that the human TRAF1 mRNA has an unusually long 5’-UTR that contains an IRES regulating TRAF1 protein translation. By performing gene transfection and reporter assays, we revealed that this IRES sequence is located within the 572 nucleotides upstream from the AUG start codon. An element between nucleotides −392 to −322 was essential for the IRES activity. Interestingly, we found that the TRAF1 expression is induced in cancer cells by chemotherapeutic drug vincristine that regulates cytoplasmic localization of PTB protein, which may contribute to the IRES-dependent translation of TRAF1 during vincristine treatment. These results indicate that TRAF1 translation is initiated via the IRES and regulated by vincristine, and suggest that regulation of the IRES-dependent translation of TRAF1 may be involved in effecting the cancer cell response to vincristine treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3154.
Abstract 3166: Inspecting the presence of sialic acid α-2-3- galactose/GalNAC terminal sugar in human stomach adenocarcinoma by lectin histochemistry
Authors: Gohari Minoo
Published: February 2011

Introduction: Glycosilation is the most frequent chemical change related to tumorogenesis which plays an important role in metastatic behavior of tumoral cells. Results of research have shown that changes in terminal sugar of glycoconjugates appear in the process of cancerization. Lectins are specific glycoproteins existing in plant and animal cells which specially attach to terminal sugar of glycoconjugates. This property of lectins make them significant markers in studies relating to histochemistry, biochemistry, recognition and differentiation of cancerous cells. The aim of this study is inspecting the presence of sialic acid α-2-3- galactose/GalNAC terminal sugar in different degrees of differentiation in human stomach adenocarcinoma by using MAA lectin in order to compare the amount of presenting this molecule in different stages and its relation with tumor progression and also for finding useful diagnostic and therapeutic method.

Methods: Tissue samples of 40 stomach adenocarcinoma with different differentiation (well, moderately, poorly and undifferentiated) accompanying with normal tissues were selected from Pathology Department of Imam Reza Hospital in Mashhad, Iran and after studing H&E samples for assuring previous diagnosis, 5micrometer sections were prepared and were stained with MAA/Alcian blue pH 2.5 and then were considered microscopic inspection.

Results: Microscopic study of samples showed no reaction in different degrees of stomach adenocarcinoma that indicates no presentence of sialic acid α-2-3- galactose/GalNAC terminal sugar, and the only positive reaction was between red blood cells of tissue vessels and MAA lectin.

Conclusion: sialic acid α-2-3- galactose/GalNAC terminal sugar is not probably present in adult stomach tissue and is not produced in the changes that result from cancerization of cells, so it probably doesn't have an important and fundamental role in cellular interactions and in making special behaviors that result from neoplasic changes in gastric gland cells.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3166.
Abstract 3167: Estrogenic regulation of S6 kinase 1 expression creates a positive feed-forward loop in control of breast cancer cell proliferation
Authors: Holz Marina K. , Seligman Fannie , Spiegel Tirtza , Maruani Denise
Published: February 2011

The 40S ribosomal S6 kinase 1 (S6K1) acts downstream of the mammalian Target of Rapamycin (mTOR), and is sensitive to inhibition by rapamycin. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types is not well understood. We have previously reported that S6K1 regulates growth of breast cancer cells by phosphorylating Estrogen Receptor α (ERα), and co-expression of S6K1 and ERα in breast cancer cells renders them sensitive to combination therapy with rapamycin and tamoxifen. In this study, we demonstrate that in response to estrogen activation, ERα activates expression of S6K1 by upregulating transcription of the RPS6KB1 gene. Increased levels of S6K1 promote further phosphorylation and activation of ERα, creating a positive feed-forward loop. We hypothesize that this relationship promotes breast cancer cell growth, thus providing a selective advantage to S6K1- and ERα-overexpressing cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3167.
Abstract 318: NAD(P)H: quinone oxidoreductase 1 possesses a strong regulatory role in MAPK signaling pathways
Authors: Patrick Brad A. , Kole Labanyamoy , Jaiswal Anil K.
Published: February 2011

NAD(P)H: quinone oxidoreductase (NQO1) is a cytosolic redox-regulating enzyme modulated by Nrf2-mediated stress signaling pathways, with an established role in regulation of stress-related transcription factors. In these studies we investigate the role of NQO1 in regulation of mitogen-activated protein kinase (MAPK) signaling pathways. Quantitative PCR and western blot studies showed that constitutive levels of several MAPK members are upregulated in NQO1-null mouse skin as well as in skin-derived keratinocytes, relative to wild type. However, though total protein levels are increased, inducibility of these kinases by known MAPK inducers such as EGF and benzo[a]pyrene (BP) is significantly decreased in NQO1-null cells. NQO1 regulation of MAPK signaling was confirmed by studies showing that NQO1 knockdown by siRNA led to decreased MAPK activation, and overexpression of NQO1 protein led to increased MAPK activation by EGF. Similarly, downregulation of activity via dicoumarol caused loss of ERK phosphorylation, while NQO1 activation via t-BHQ caused increased ERK phosphorylation, which was blocked by MAPK pathway-specific inhibitors. To investigate the functional role of MAPK deactivation in skin-derived keratinocytes, studies with BP and menadione showed that lack of NQO1 expression led to increased susceptibility to ROS generation, and that this ROS generation causes increased toxicity. Taken together, these results show a significant role for NQO1 in regulation of MAPK signaling pathways, which ultimately leads to protection against ROS-mediated apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 318.
Abstract 3180: Altered β-catenin phosphorylation pattern in human prostate cancer
Authors: Du Cheng , Zhang Chuanyou , Biswas Md. Helal U. , Balaji K.C.
Published: February 2011

Protein kinase D1 (PKD1) phosphorylates β-catenin at Thr112 and Thr120. The phosphorylation is required for β-catenin membrane trafficking via trans Golgi network (TGN). Data presented in this study indicate that activation of PKD1 shrinks the cytoplasmic pool of β-catenin which mainly is the non-phosphorylation (S33/S37/T41/S45) isoform of β-catenin, suggesting that PKD1 interacts with Wnt/GSK3β signaling pathway. The phosphorylated Thr120 β-catenin (pT120) isoform predominately locates at membrane fraction in cultured prostate cancer cell line C4-2. A newly developed phospho-threonine antibody specifically targeting the pT120 of β-catenin shows accumulation of pT120 β-catenin in TGN in normal prostate tissues. Our study demonstrates that distribution of Thr120 phosphorylated β-catenin is distinct in normal and malignant prostate tissues. In 16/22 (72%) of normal prostate tissues, the pT120 β-catenin is enriched at TGN. In contrast, only 15/200 (7.5%; p < 0.001) of prostate cancer samples had accumulation of pT120 β-catenin in TGN. A vast majority of cancer samples (92.5%) lacking of TGN staining shows the pT120 β-catenin distributed diffusely in cytoplasm, nucleus and plasma membrane. can be more discriminatory compared to conventional β-catenin antibody staining. We propose that β-catenin T120 phosphospecific antibody can be used to study alteration in subcellular distribution of β-catenin and possibly as biomarker in prostate and other tissues, which may facilitate risk stratification or predict disease outcomes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3180.
Abstract 3191: Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating hedgehog signaling
Authors: Li Fuchuan E. , Shi Wen , Capurro Mariana , Filmus Jorge
Published: February 2011

Glypican-5 is one of the six members of the glypican family. It has been previously reported that glypican-5 stimulates the proliferation of rhabdomyosarcoma cells. In this study we show that this stimulatory activity of glypican-5 is due to its ability to promote Hedgehog signaling. We have previously shown that glypican-3, another member of the glypican family, inhibits Hedgehog signaling by competing with Patched for Hedgehog binding. Furthermore, we showed that glypican-3 binds to Hedgehog through its core protein but not to Patched. Here we demonstrate that glypican-5 can increase the binding of Sonic Hedgehog to Patched. We also show that glypican-5 can bind to both Hedgehog and Patched through its glycosaminoglycan chains. Interestingly, we found that the heparan sulfate chains of glypican-5 display a significantly higher degree of sulfation than those from glypican-3. In addition, we show that glypican-5 interacts with Hedgehog with a lower affinity than glypican-3. Based on these results, we propose that glypican-5 stimulates Hedgehog signaling by facilitating/stabilizing the interaction between Hedgehog and Patched.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3191.
Abstract 3202: Imetelstat inhibits telomerase activities in xenograft tumors and bone marrow cells and inhibits tumor growth in xenograft models at plasma exposures equivalent to current clinical exposures in Phase I trials
Authors: Go Ning F. , Villarreal Lyssa , Pattamata Preeti , Nazzal Denise , Lin Tong , Khor Soo-Peang , Delavan-Boorsma Karen , Tressler Robert
Published: February 2011

Telomerase activation is a hallmark of cancer and is critical for dysregulated cell proliferation and cell immortality. Increased telomerase activities were detected in essentially all epithelium derived cancers tested up-to-date and in many hematological malignancies as well, such as multiple myeloma and leukemia. Levels of telomerase activities in the malignant tissues are significantly higher compared to those detected in the normal, regenerating somatic tissues, such as liver and bone marrow cells. Given the differential activation states of telomerase in malignant and normal tissues, telomerase is considered an attractive cancer target by controlling telomere extension and cell immortality. Imetelstat is a first-in-class, lipidated oligonucleotide thio-phosphoramidate telomerase inhibitor. It is a potent and specific telomerase RNA template antagonist, currently in phase I clinical development. Objectives of this study were to demonstrate the in vivo telomerase activity inhibition in xenograft tumors and in bone marrow tissue in animal models, and to correlate the telomerase inhibition effect with mouse plasma drug exposure and xenograft tumor growth inhibition. Our preliminary results showed that treatment with imetelstat at 15-30 mg/kg tiw resulted in plasma concentrations greater than the IC50 for in vitro telomerase inhibition and a plasma exposure level comparable to the tolerable exposure in the clinic. Imetelstat treatment at 15-30 mg/kg tiw for 6 weeks inhibited OVCAR-5 xenograft tumor growth in vivo. Moreover, telomerase inhibition in the xenograft tumors and in the mouse bone marrow was demonstrated. Our results provided evidence of the specific target inhibition by imetelstat treatment in an in vivo system. Our data also suggested a pharmacodynamically efficacious dose level may have been achieved in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3202.
Abstract 3205: Identification of senescence-inducing microRNAs in normal human epithelial cells
Authors: Shin Ki-Hyuk , Pucar Ana , Kim Reuben , Kang Mo , Park No-Hee
Published: February 2011

MicroRNAs (miRNAs) are novel epigenetic regulators of eukaryotic gene expression and play key roles in many cellular processes. However, the contribution of miRNAs to replicative senescence remains largely unexplored. To understand the role of miRNA in replicative senescence of normal human epithelial cells, we examined the expression profiles of 847 miRNAs in non-senescent (young) and senescent (old) normal human oral keratinocytes (NHOKs). We identified 126 senescence-associated miRNAs (SA-miRs) whose expression was altered greater than 2-fold with senescence. Among SA-miRs, 117 miRNAs (93%) were up-regulated and 9 miRNAs (7%) were down-regulated in senescent NHOKs compared to non-senescent NHOKs. miR-137 and miR-668 were consistently up-regulated with senescence of three independent NHOK cultures and with ionizing irradiation, a premature senescence inducer of NHOKs. Overexpression of miR-137 and miR-668 significantly increased senescence-associated β-galactosidase (SA β-gal) activity and senescence markers, p16INK4A and p53 in non-senescing NHOKs, indicating that they are senescence-inducing miRNAs. Furthermore, we found that these senescence-inducing miRNAs were frequently downregulated in human head and neck squamous cell carcinoma cell lines. Since senescence is considered as a tumor suppressor mechanism, the newly identified senescence-inducing miRNAs need to be considered for therapeutic application for cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3205.
Abstract 3210: pRb, but not p107 or p130, is required for full induction of p16INK4a in human mammary epithelial cells
Authors: Bazarov Alexey , Korobochkina Irina , Bosire Moses , Hines Curtis , Mukhopadhyay Rituparna , Lee Won Jae , Chicas Agustin , Lowe Scott , Yaswen Paul
Published: February 2011

Pocket family proteins pRb, p107 and p130 are important cell cycle regulators in mammalian cells. In addition to controlling G1 to S transition, they also participate in differentiation and senescence. While all three pocket proteins have similar structures and apparently overlapping functions, defects in only one, pRb, are associated with human cancers. In breast cancer, pRb loss is associated with triple negative (ER-, PR-, HER2-) tumors, which tend to be more aggressive. To explore possible functions of pRb that may be unique, we investigated relative contributions of pocket proteins to cellular senescence initiated by high levels of the cyclin-dependent kinase inhibitor p16INK4a. For this purpose, we silenced pRb, p107 and p130 expression individually with shRNA constructs and exposed MCF7 and MDA-MB-231 breast cancer cell lines to tet-inducible p16. In both malignant lines, knockdown of any single pocket protein was insufficient to prevent p16-induced senescence, indicating the likely existence of compensatory activities of the three pocket proteins in mediating p16-induced senescence. However, our finding that the introduction of HPV E7 - which inactivates all three pocket proteins, prevented p16-induced senescence, is consistent with the hypothesis that as a group, pRb, p107 and p130 constitute essential mediators of p16-induced senescence. To examine the relative contributions of the pocket proteins to cellular senescence initiated by endogenous p16, we used cultured primary human mammary epithelial cells (HMEC) from non-malignant breast tissues. These cells spontaneously induce endogenous p16 and undergo senescence. When we examined the relative contributions of pocket proteins to senescence associated with increased endogenous p16 levels in HMEC, we found that suppressing pRb alone abrogated p16-induced senescence, whereas suppressing p107 or p130 did not. Remarkably, HMEC in which pRb alone was knocked down showed reduced p16 expression and longer proliferative life spans than controls or HMEC in which p107 or p130 were knocked down. These results suggest that pRb, but not p107 or p130, is required for full induction of endogenous p16 in breast cells, and provide a possible explanation for the special tumor-suppressor role of pRb.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3210.
Abstract 3211: PTTG1 attenuates drug-induced cellular senescence
Authors: Tong Yunguang , Zhao Weijiang , Wawrowsky Kolja , Melmed Shlomo
Published: February 2011

PTTG1 (pituitary tumor transforming gene) is abundantly expressed in several cancers and levels correlate with invasiveness and clinical outcome. Here we show that colon cancer cells devoid of PTTG1 (PTTG1−/−) demonstrate enhanced sensitivity and development of senescence in response to anti-neoplastic drugs including doxorubicin and trichostatin A (TSA). Doxorubicin (0.005 uM) had no effect on WT HCT116 colon cancer cells but inhibited PTTG1−/− HCT116 cell BrdU incorporation by 37 % (p<0.001). β-Galactosidase staining demonstrated that 43 % of PTTG1−/− HCT116 cells were senescent compared to 3 % of WT cells treated with 0.005 uM doxorubicin. Up to 0.02 uM doxorubicin did not cause apoptosis as measured by Tunel assay and activated caspase 3 levels, suggesting that cell cycle arrest is responsible for enhanced drug sensitivity in PTTG1−/− cells. PTTG1−/− cells exhibit ∼3 fold induction of p21 upon doxorubicin or TSA treatment, while knock down of p21 abrogated senescent effects of these drugs. p21 expression in PTTG1−/− cells after doxorubicin and TSA treatment was increased by enhanced binding of Sp1, p53 and p300 at the p21 promoter. PTTG1−/− cells developed smaller tumors than WT (1.24 ± 0.41 vs 0.39 ± 0.15 g, p<0.001), and were also more sensitive to doxorubicin treatments, with tumor growth inhibition rate 50% and 75 % respectively (p<0.05). PTTG1−/− cells develop less metastatic foci compared to WT (29 ± 8 vs 46 ± 12, p<0.001). Doxorubicin treatment suppressed metastatic foci formation by 49 % in PTTG1−/− tumors, compared to 15 % in WT cell-derived tumors. β-Galactosidase staining indicated that cell senescence was more pronounced in doxorubicin-treated PTTG1−/− tumors than in WT (57 % vs 22 % in subcutaneous tumor, p<0.001, 70 % vs 18 % in metastatic foci, p<0.001,). As senescence is a determinant of the cell response to anti-neoplastic treatments, the results indicate that PTTG1 may decrease anti-neoplastic drug responsiveness through attenuating cell senescence.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3211.
Abstract 3218: Bisphosphonates induce premature senescence and impair proliferation and migration of normal human oral keratinocytes
Authors: Kim Reuben H. , Lee Rachel , Woo Jessica , Dong Qing-Hua , Shin Ki-Hyuk , Kang Mo , Park No-Hee
Published: February 2011

Bisphosphonates (BPs) are potent anti-resorptive agents that are widely used to treat metastatic bone diseases in cancer patients. BPs target the mevalonate pathway and inhibit cholesterol synthesis and prenylation (e.g., geranylgernaylation and farnesylation) of small GTPases that are important for cell proliferation and survival. Long-term users of BPs are at the higher risk of developing osteonecrosis of the jaw (ONJ), which commonly occurs at the site of previous tooth extraction or other surgical interventions. Although bisphosphonate-related osteonecrosis of the jaw (BRONJ) is clinically defined as exposed necrotic bone with unhealed and open oral mucosa, the exact role of oral mucosa in the pathophysiology of BRONJ is not fully understood. The current study was undertaken to examine direct effects of BPs on the reepithelialization of oral mucosal cells. When normal human oral keratinocytes (NHOK) were treated with pamidronate (PAM), proliferation and migration were inhibited in dose-dependent manner as determined by MTT assay, proliferation assay, Western blotting against PCNA, and the scratch wound healing assay. Cell cycle analysis revealed increased S-phase content in PAM-treated NHOK, and this was associated with decreased mRNA and protein expressions of Cyclin A2 but not Cyclin D. PAM also induced premature senescence in NHOK as determined by increased β-galactosidase positivity, increased p16 expression, and phosphorylated p-38 MAP kinase pathway. PAM-induced premature senescence and proliferation inhibition were partially reversed by geranylgerniol (GGOH), but not farnesol (FOH), which reconstitute geranylgeranylation and farnesylation of the mevalonate pathway, respectively. Finally, using 3-dimensional (3D) raft culture system, we found that PAM-treated oral mucosal tissue constructs showed diminished immunohistochemical staining of PCNA and loss of reepithelialization in the wounded area, indicating that BPs impair proliferation and migration of oral epithelium constructs ex vivo. Our study demonstrates that BPs induce premature senescence and directly impair proliferation and migration of oral mucosal cells, which may explain the pathophysiology of BRONJ in cancer patients undergoing BP-treatment. This study was supported by NIDCR/NIH (DE017121).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3218.