bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1059: Neuroendocrine-derived peptides stimulate IGF-1R signaling to promote prostate cancer cell survival
Authors: DaSilva John O. , Amorino George P. , Casarez Eli V. , Pemberton Bradley , Parsons Sarah J.
Published: February 2011

A common feature of the most aggressive hormone-refractory prostate cancers is the accumulation of cells with neuroendocrine characteristics that produce paracrine growth factors, and may provide a proliferative advantage to cancer cells during disease progression. We have previously shown that neuroendocrine-derived peptides transactivate EGFR and Src signaling to promote growth of prostate cancer cells under low concentrations of androgen. In this study we demonstrate that in addition to the transactivation of EGFR family of receptors, neuroendocrine-derived factors enhance IGF-1R signaling through the transcriptional regulation of downstream components of the pathway to promote cell survival.

cDNA microarray analysis to compare the gene expression profile of LNCaP cells in the presence or absence of neuroendocrine-derived conditioned medium (NE-CM) revealed differential regulation of multiple IGF-1R pathway signaling components including the receptor protein tyrosine phosphatase (PTP-RF) and Akt. Quantitative RT-PCR confirmed the downregulation of PTP-RF upon treatment of LNCaP cells with NE-CM for 24 hours that was associated with a reduction in protein levels. IGF1-R and Akt phosphorylation levels were enhanced and sustained over the 24 hour period. Statistically significant changes in cell survival, cell death, and apoptosis were observed with various experimental approaches for both LNCaP and PC3 cells treated with NE-CM. In addition, NE-CM was able to protect the prostate cancer cells from docetaxel-induced apoptosis. The pro-survival effects of NE-CM were abrogated in part by specific antagonists of the neuropeptides neurotensin, bombesin, and PTHrP, as well as selective inhibitors of IGF-1R, EGFR and Src. These data support the hypothesis that neuroendocrine-like cells secrete factors that act upon PC cells to stimulate pro-survival signaling mediated in part by IGF-1R, EGFR, and c-Src.

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 1059.
Abstract 1061A: Role of protein tyrosine phosphatase PTPH1 in breast cancer
Authors: Zhi Huiying , Hou Song-wang , Li Rong-shan , Basir Zainab , Welsh Joellen , Chen Guan
Published: February 2011

Tyrosine phosphorylation, which is controlled by the balanced action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), regulates critical cellular processes including proliferation and malignant transformation. While PTKs have a well-established role in breast cancer, the contribution of PTPs remains mostly unknown. Our recent studies demonstrated that the PTPH1 tyrosine phosphatase promotes K-Ras-dependent colon cancer growth through binding and dephosphorylating p38γ MAPK, and here we showed that PTPH1 increases breast cancer growth through the nuclear receptor VDR (vitamin D receptor). PTPH1 was shown to be over-expressed in about 50% of primary breast tumors and importantly its expression levels are significantly higher in HER-2 positive breast cancers. PTPH-forced expression increases breast cancer growth independent of its phosphatase activity and its oncogenic activity was further established by a growth inhibitory response through shRNA-mediated PTPH1 depletion. Mechanistically, PTPH1 was found to specifically induce the nuclear receptor VDR but not ER or PR expression and inhibition of endogenous VDR expression by shRNA or the genetic knockout completely blocks PTPH1-induced growth stimulation, which couples with an altered intracellular VDR localization. These results reveal a promoting role of a novel tyrosine phosphtase PTPH1 in breast cancer growth through signaling cross-talk with VDR, which may be a new target for breast cancer control.

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 1061A.
Abstract 1064: Numb, a progenitor cell fate determinant and intrinsic inhibitor of Notch signaling, is cell cycle regulated and required for proper mitotic entry and progression in melanoma cells
Authors: Schmit Travis L. , Setaluri Vijayasaradhi , Spiegelman Vladimir S. , Ahmad Nihal
Published: February 2011

Proper regulation of the cell cycle, particularly DNA replication and cell division, are crucial for correct cell ploidy and genomic integrity. Loss of various regulators, often tumor suppressors, during these processes leads to either cell cycle arrest or unregulated progression through mitosis. This may result in aneuploidy and neoplastic transformation. Therefore, defining the mechanisms by which various tumor suppressors exert their regulatory functions is essential for understanding cancer development as well as developing novel target-based therapies to combat cancer progression. We found that Numb expression is cell cycle regulated and that its expression is required for proper mitotic entry and exit in melanoma cells. Using a double thymidine arrest and release protocol, we evaluated the expression of Numb during the cell cycle. Numb mRNA and protein expression was found to be maintained at a low steady state level in asynchronous A375 and HS294T melanoma cells. However, as cells entered S phase Numb levels were found to increase, peak during mitosis and then rapidly decline at mitotic exit when the cells reentered G1 Phase. This cycling of Numb mirrored the mitotic specific expression of both Plk1 and CyclinB1, however Numb showed an expression pattern with an increase beginning slightly earlier (as early as S phase). Plk1 inhibition (via shRNA as well as a small molecule inhibitor) and nocodazole-mediated mitotic arrest was also found to be accompanied with an increase in Numb expression. Additionally, we found that Plk1 and Numb co-immunoprecipitated and Plk1 was capable of phosphorylating Numb indicating a possible regulation of Numb by Plk1. Further, surprisingly, we found that lentiviral shRNA mediated knockdown of Numb resulted in a G2/M cell cycle arrest (using FACS analysis) and a significant reduction in cell growth (using Trypan blue exclusion) in both melanoma cell lines. Overall, our observations suggests that Numb is a regulator of proper cell cycle progression and may possess tumor suppressor functions.

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 1064.
Abstract 1067: FOXD3 is a B-RAFV600E-regulated inhibitor of G1/S progression in melanoma cells
Authors: Abel Ethan V. , Aplin Andrew E.
Published: February 2011

The forkhead box transcription factor FOXD3 is a stemness factor that prevents the production of melanocyte progenitors from the developing neural crest; however, its role in human cancers is not known. Here we demonstrate that FOXD3 levels are up-regulated following attenuation of B-RAF and MEK signaling in mutant B-RAF harboring human melanoma cells. This effect was selective since FOXD3 was not up-regulated following MEK inhibition in wild-type B-RAF melanoma cells and mutant B-RAF thyroid carcinoma cells. Ectopic FOXD3 expression potently inhibited melanoma cell growth without altering mutant B-RAF activation of ERK1/2. Inhibition of cell growth was due to a potent G1 cell cycle arrest that was largely dependent on p53 and, to a lesser extent, its downstream target p21Cip1. These studies demonstrate that FOXD3 is suppressed by B-RAF, uncover a novel role and mechanism for FOXD3 as a negative cell cycle regulator and have implications for the repression of melanocytic lineage 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 1067.
Abstract 1069: The role of mammalian Ecdysoneless in cell cycle regulation and cancer
Authors: Gurumurthy Channabasavaiah B. , Kim Jun H. , Zhao Xiangshan , Naramura Mayumi , Band Hamid , Band Vimla
Published: February 2011

Our long term research goal is to understand biochemical mechanisms of immortalization of human mammary epithelial cells (hMECs) that represents an early step in breast cancer progression. We have generated several hMEC immortalization models using viral oncogenes, mutated cancer genes as well as radiation treatment of cells.

In one of such immortalization model, we identified Ecdysoneless (Ecd) protein as the human papilloma virus (HPV) E6 interacting protein. We have reported earlier that HPV16 E6 protein is highly efficient for hMEC immortalization. Here, we present evidence that Ecd, an evolutionarily conserved protein, is a novel regulator of the Rb/E2F pathway. We generated a conditional Ecd knockout mouse model and observed that complete deletion of Ecd leads to early embryonic lethality in mice. The experiments using conditional Ecd-null MEFs created from Ecdlox/lox revealed that lack of Ecd inhibits cellular proliferation by delaying G1-S cell cycle progression and down-regulates E2F target gene expression. Furthermore, Ecd directly binds to Rb at the pocket domain and disrupts the interaction of Rb with E2F, leading to impairment of repressive effect of Rb on E2F target promoters. Thus, we demonstrate that Ecd is an essential protein in embryonic development and a novel regulator of G1-S cell cycle transition by modulating Rb/E2F pathway. Consistent with these observations, overexpression of Ecd in hMECs hastens the exit of cells from G1 phase of the cell cycle. Significantly, immunohistochemical analyses showed Ecd is overexessed in ductal carcinoma in situ as well as in infiltrating ductal carcinoma of breast. In conclusion, Ecd plays an important role in cell cycle progression and its deregulation can lead to abnormal cell proliferation leading to breast cancer 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 1069.
Abstract 1070: Circadian transcriptional factor Period 2 modulates cyclin B1 expression
Authors: Yang Jianhua , Finkielstein Carla V.
Published: February 2011

The proper timing of cell division is a major factor contributing to the regulation of normal growth and emerges as a fundamental process in the development of most cancers. The existence in most, if not all, living organisms of many behavioral and physiological events that occur at well-defined and controlled times argues for the presence of time-keeping systems that allow them to adapt to cyclic changes in environmental conditions. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression. In the present study, we use cDNA microarray analysis to identify Per2 target genes following siRNA knock-down expression of Per2 in mouse hepatocytes AML12 cells. We identified cyclin B1, a key G2/M regulator of the cell cycle, as a downstream target gene that is negatively regulated by Per2 under normal conditions. Quantitative real-time PCR confirmed our observations by showing cyclin B1 expression is enhanced under Per2 knock-down condition. Accordingly, Bmal1 expression, a target gene of Per2, was specifically augmented under Per2 over-expression conditions. Further experiments focused in characterizing the regulation of the human cyclin B1 promoter by Per2 using luciferase reporter assays. Results show cyclin B1 reporter activity is enhanced in siPer2 transfected cells compared to control scrambled siRNA. Promoter analysis defined the region comprising −484 to −284 of human cyclin B1 promoter containing response elements for gene trans-activation while the region between −284-1 is mainly responsible for Per2 regulation. Our results show the canonical E-box between −284-1 is not required for Per2 regulation of cyclin B1, suggesting non-canonical E-boxes might be directly responsible for Per2-mediated expression. FACS analysis revealed that siPer2 treated cells exhibit a reduced G2/M phase with correspondingly higher cyclin B1 protein levels and reduced Tyr-15-cdc2 phosphorylation compared to scrambled siRNA treated cells. Our previous results show Per2 stability is directly controlled by the endogenous levels of heme whose endogenous levels oscillate in a 24 h cycle. Here, we evaluated whether cyclin B1 expression is altered in heme-treated cells. In contrast to its up-regulation in siPer2 treated cells, luciferase reporter assay showed a decrease in cyclin B1 promoter activity in response to heme treatment. These results may reflect a complex pattern of regulation in which Per2 acts as a sensor molecule to monitor changes in both environmental and cellular conditions that will impact cell 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 1070.
Abstract 1075: Cul1 Expression Is Increased in Early Stages of Human Melanoma
Authors: Chen Guangdi , Cheng Yabin , Martinka Magdalena , Li Gang
Published: February 2011

As the largest family of ubiquitin-protein E3 ligases, the SCF (Skp1/Cullin/Rbx1/F-box protein) complexes ubiquitinate a broad range of proteins involved in cell cycle progression, signal transduction and transcription. Cullin1 (Cul1) serves as a rigid scaffold in SCF complex for Rbx1, Skp1 and F-box protein subunits assembly and aberrant expression of Cul1 is involved in dysfunction of SCF E3 ligases. To investigate the role of Cul1 in the development of melanoma, we examined the expression of Cul1 in melanocytic lesions at different stages and analyzed the correlation between Cul1 expression and clinicopathologic parameters by tissue microarray and immunohistochemistry. The result showed that Cul1 expression was significant increased in primary and metastatic melanoma compared with dysplastic nevi, while there was no significant difference of Cul1 expression between primary and metastatic melanoma, which suggested that Cul1 plays an important role in the initiation stage of melanoma development. We found knockdown of Cul1 inhibited melanoma cell growth and overexpression of Cul1 promoted melanoma cell growth through regulating CDK inhibitor p27Kip expression. Knockdown of Cul1 abrogated Skp2-induced p27 degradation in melanoma cells. These results suggested that Cul1 is involved in cell cycle regulation of melanoma cells via Cul1-dependent ubiquitination and degradation of the p27kip1 by SCFSkp2 complex. In conclusion, our data indicate that Cul1 may serve as a potential marker for human melanoma initiation and early diagnosis.

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 1075.
Abstract 1076: Reversible effect of all-trans-retinoic acid on AML12 hepatocyte proliferation and cell cycle progression
Authors: Murphy Lynea , Moore Tanya , Nesnow Stephen
Published: February 2011

The role of all-trans-retinoic acid (atRA) in the regulation of cellular proliferation and differentiation is well documented. Numerous studies have established the cancer preventive properties of atRA which functions to regulate levels of cell cycle proteins essential for the G1/S transition and progression through S phase. Using AML12 cells, a non-transformed immortalized mouse hepatocyte cell line, the goals of this study were to first determine if changes in AML12 cell proliferation could be observed in response to atRA treatment and establish whether this was a suitable system to investigate molecular mechanisms altered by hepatocarcinogens. Cells were treated with 1µM and 5 µM atRA for 2 to 10 days. At various time points, control (DMSO) and treated cells were enumerated to determine the effect of atRA on AML12 cell proliferation. At 5 days of atRA treatment, there was a 56% decrease in the number of atRA treated cells when compared to control cells. This effect was reversible as an increase in cell number was observed two days after removal of atRA from the treatment media. Cell cycle analysis by flow cytometry using propidium iodide stained AML12 cells showed a significant reduction (p<0.05) in S phase cells and a significant increase (p<0.05) in G1 phase cells with 48 hours of atRA treatment when compared to control cells indicating a block at the G1/S checkpoint. Western analysis of control and treated whole cell lysates revealed a decrease in phosphorylated (Serine807/Serine811) retinoblastoma protein (pRb). Taken together, these results indicate that proliferation in AML12 hepatocytes can be reversibly modified by atRA by regulating these cells at the G1/S checkpoint with a reduction in pRb. Previous studies of liver tissues collected from mice exposed to the hepatocarcinogenic fungicide, propiconazole, have revealed an increase in cellular proliferation, increases in atRA metabolism as well as decreases in the levels of hepatic atRA. Based on the results of the present experiments, AML12 hepatocytes provide an optimal in vitro system to elucidate the molecular mechanism by which propiconazole induces hepatotumorigenesis. This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy.

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 1076.
Abstract 1077: Galectin-1 is implicated in G1/S-checkpoint control following irradiation - possible explanation of Galectin-1 mediated radioresistance
Authors: Huebner Alexander , Perry James , Siedow Michael , Zhang Min , Chakravarti Arnab , Lautenschlaeger Tim
Published: February 2011

Purpose:

The purpose of the conducted study was to examine a possible therapeutic relevance of Galectin 1 (Gal-1), a beta-galactoside binding protein. Gal-1 is overexpressed in high grade gliomas and leads to increased radioresistance in glioma cell culture models. Inhibition of Gal-1 increases radiosensitivity and thus is a potential therapeutic target for radiation sensitization. P53 has been shown to negatively regulate Gal-1, hence radiosensitivity could possibly be cell cycle mediated.

Experimental Design:

We used flow cytometry based cell cycle analysis to investigate cell cycle distribution of LN 229 glioma cells following irradiation. LN229 control cell lines and shRNA mediated Gal-1 knockdown LN 229 cell line were exposed to 6 Gy of irradiation and cell cycle analysis was performed. Furthermore Western blot analysis was carried out, probing for multiple proteins implicated in cell proliferation. The phosphorylation status of a variety of phosphorylation sites of p53, Chk1/2 and AKT were examined. Additionally p53-Gal-1 double-knockdown cell lines were created and clonogenic survival assays as well as AnnexinV apoptosis assays were conducted to analyze the influence on radiation sensitivity.

Results:

The results of the flow cytometry based cell cycle analysis show significant differences between the LN 229 control cell line and the Gal-1 knockdown cell line after irradiation. 24 h after irradiation 77 % of the control line cells are in G1 whereas only 60 % of the Gal-1 knockdown cells are in G1.

Previous data has identified differences in the phosphorylation status of Akt in control vs. Gal-1 knockdown glioma cell lines. Phosphorylation of Akt is induced after irradiation. Western blot analysis indicates a reduction of phosphorylated Chk1/2 in Gal1 knockdown cell lines after irradiation. The phosphorylation state of the p53 phosphorylation site, correlating with Chk1/2 activity, exhibit anticipated results.

P53-Gal-1 double-knockdown cell lines demonstrate significantly reduced clonogenic survival compared to control cell lines.

Conclusions:

The finding that less Gal-1 knockdown cells are in G1 phase after irradiation and that there is a reduction in phosphorylated Chk1/2 leads to the conclusion that Gal-1 knockdown abrogates G1 arrest through a Chk1/2 mediated pathway. That means in reverse that Gal-1 can mediate cell cycle arrest, thus potentially can explain Gal-1 mediated radioresistance. Further analysis of Chk1/2 independent phosphorylation sites of p53 is going to be conducted.

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 1077.
Abstract 1081: The role of calcium in relationship of microtubule stabilization, mitotic arrest, and apoptosis induced by Taxol in breast cancer cells
Authors: Pan Zhi , Gollahon Lauren
Published: February 2011

Background: Our previous study showed that Taxol, an important anticancer agent for breast cancer, directly releases calcium from the endoplasmic reticulum (ER), promoting subsequent apoptosis in breast cancer cells. Taxol also stabilizes microtubules, causing mitotic arrest. The relationship between these events and apoptosis remains unclear. The purpose of this study was to investigate the connection between calcium regulation, mitotic arrest and apoptosis induced by Taxol treatment.

Objective & Specific Aims: To accomplish this, we (1) Visualized the microtubule changes induced by Taxol at different dosages after different exposure times, (2) Quantified the cell cycle arrest induced by Taxol at different dosages after different exposure times, (3) Analyzed whether agents that interfere with intracellular calcium fluxes affect microtubule stabilization, mitotic arrest and apoptosis during Taxol treatment.

Study Method: Breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 were treated with two Taxol dosages (∼10−7M and ∼10−6M). Microtubule changes were detected using immunocytochemistry. Cell cycle analysis was detected using flow cytometry. Cells were pretreated with the cytosolic calcium chelator BAPTA-AM and the ER calcium inhibitor 2-APB to interfere with the calcium changes induced by Taxol. This allowed us to determine whether Taxol-induced mitotic arrest was calcium dependent. Free cytosolic calcium changes were measured by Fluo-AM calcium dye, and ER calcium changes were specifically measured by the advanced calcium cameleon D1ER.

Results & Relevance: Our data showed that microtubule stabilization and mitotic arrest were observed (6h) before significant apoptosis occurred at 12h exposure. Pretreatment of calcium agents inhibited both mitotic arrest and apoptosis induced by Taxol. High (∼10−6M) dosage Taxol induced calcium-promoting mitotic arrest, whereas low (∼10−7M) dosage Taxol did not. A similar pattern was also observed in our previous apoptosis analysis, indicating calcium, cell cycle arrest, and apoptosis are tightly associated with each other and need further investigation. In summary, our results suggest elucidating the role of calcium is essential to understand the process through which the widely used anticancer agent Taxol induces mitotic arrest and apoptosis. It will provide new insights into the action of Taxol and aid in the development of more effective breast cancer therapies.

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 1081.
Abstract 1082: BI 811283, a potent inhibitor of the mitotic kinase Aurora B, shows dose- and schedule-dependent efficacy in human cancer xenograft models
Authors: Gürtler Ulrich , Tontsch-Grunt Ulrike , Zahn Stephan K. , Quant Jens , Adolf Guenther R. , Solca Flavio
Published: February 2011

Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. The essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for therapeutic intervention. BI 811283, a potent inhibitor of this key mitotic regulator, inhibits proliferation of a wide range of cultured human cancer cells at low nanomolar concentrations by inducing polyploidy, senescence and apoptosis.

Methods. BomTac:NMRI-Foxn1nu mice were grafted subcutaneously with NCI-H460 non-small cell lung carcinoma (mutant KRAS, wild-type p53), HCT 116 colon carcinoma (mutant KRAS, wild-type p53) or BxPC-3 pancreas adenocarcinoma cells (wild-type KRAS, mutant p53). Treatment was initiated when the tumors had reached a volume of ∼50 mm3. BI 811283 was injected intravenously once or twice weekly as a single bolus or b.i.d. Alternatively, the compound was administered once-weekly by a continuous 24 h infusion via subcutaneously implanted osmotic mini-pumps. Multiple dose levels and dosing schedules were evaluated.

Results. In models of human non-small cell lung cancer, colon carcinoma and pancreas carcinoma, multiple cycles of treatment with BI 811283 at total weekly doses of 20 to 75 mg/kg resulted in dose-dependent inhibition of tumor growth or tumor regression. Continuous s.c. infusion at 20 mg/kg over 24 h once-weekly was clearly superior to all bolus injection schedules delivering weekly doses up to 75 mg/kg. Furthermore, regression of large tumors (350 mm3) was induced in the HCT 116 colon carcinoma model. Biomarker analyses of HCT 116 tumors revealed that therapeutic doses of BI 811283 inhibited phosphorylation of histone H3, a direct substrate of Aurora B. Histological examination showed an accumulation of enlarged, multinucleated cells in accordance with the expected mechanism of action.

Conclusions. BI 811283 has demonstrated potent antitumor activity in multiple cancer models at well-tolerated doses; treated tumors show hallmarks of Aurora B inhibition. Continuous infusion over 24 h provides a superior therapeutic index compared with bolus administration. The compound is currently under investigation in Phase I clinical trials.

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 1082.
Abstract 1088: Differences in p53 signaling following induction of nucleotide pool deficiencies with hydroxyurea or PALA
Authors: Heyer Cortney L. , Moran Richard G.
Published: February 2011

The tumor suppressor p53 is stabilized following the induction of deficiencies in nucleotide pool levels by antimetabolites. N-(phosphonacetyl)-L-aspartate (PALA) reversibly inhibits aspartate transcarbamylase, causing interruption of de novo pyrimidine synthesis; hydroxyurea (HU) reversibly inhibits ribonucleotide reductase, inhibiting the conversion of ribonucleotide triphosphates to deoxynucleotide triphosphates. When HCT116, a human colon carcinoma cell line, is treated with either HU or PALA, p53 accumulates and cells arrest in S-phase. Beyond these similarities, the cellular responses to PALA and HU are strikingly different. Chk1 is phosphorylated to a far greater extent in HU-treated HCT116 cells than following PALA treatment. The lack of Chk1 phosphorylation observed during PALA treatment was found to be due to the p53-dependent transcriptional activation of the Wip1 phosphatase, rather than the inactivity of ATR. Chromatin immunoprecipitation studies performed on PALA-treated cells demonstrated the presence of p53 at the Wip1 promoter, and both Wip1 transcript and protein levels were elevated. In contrast, the residency of p53 over the Wip1 promoter was not observed during HU treatment, nor was Wip1 protein or transcript elevated. During PALA treatment of the isogenic p53 null HCT116 cell line, Wip1 transcript levels are not elevated and Chk1 phosphorylation is restored, demonstrating the role of p53 in the elevation of Wip1 following PALA treatment. Although p53 accumulates during both HU and PALA treatment, the responses to the individual drugs differ so greatly that PALA-treated cells undergo a p53-dependent apoptosis while HU-treated cells enter a cytostatic cell cycle arrest. The post-translational modifications of p53 are dissimilar in the two treatments suggesting a variation of signaling to p53 as well as the downstream actions of the modified p53. The accumulated p53 in PALA-treated cells is phosphorylated at serine 46, an event needed to direct p53 to pro-apoptotic promoters; this phenomenon is not observed in HU-treated cells. Hence, during de novo pyrimidine synthesis inhibition, p53 promotes Wip1 transcription causing dephosphorylation of Chk1 leading to apoptosis. When deoxynucleotide triphosphate synthesis is inhibited, Chk1 is phosphorylated promoting cellular arrest in S-phase.

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 1088.
Abstract 1097: Arf induction by Tgf-beta2 is mediated by Smad-2/3 dependent enhancement of Arf transcription
Authors: Zheng Yanbin , Zhao Yi D. , Chu Patricia , Skapek Stephen X.
Published: February 2011

The Arf tumor suppressor gene product, p19Arf, is induced in response to certain oncogenic signals to block cancer development and progression. How the oncogenic cues are coupled to Arf promoter activation remains poorly defined, though. We took advantage of the fact that the Arf promoter is strictly controlled during development to investigate the normal regulatory mechanisms. We have recently published that Tgf-beta2 is required for Arf promoter activation at several sites in the developing mouse. To define mechanisms for the regulation, we developed a cell culture-based assay. We now show that Tgf-beta2 (5 ng/ml) induced p19Arf expression in WT mouse embryo fibroblasts (MEFs), enhanced Arf promoter activity in Arf lacZ/lacZ MEFs, and maintained a G1 cell cycle arrest in an Arf-dependent manner. The Arf promoter was also increased by Tgf-beta1, which functionally overlaps with Tgf-beta2, but not by Pdgf-B, a potent mitogen, nor BMP-2, another Tgf-beta superfamily member. To elucidate which pathway mediated the Tgf-beta2 effects, we used a panel of chemical inhibitors of Smad-dependent and -independent pathways. Among them, SB431542, a Tgf-beta type I receptor inhibitor, blocked Smad2 phosphorylation and impeded Tgf-beta2 induction of Arf. Genetic studies confirmed this because RNAi-driven knock-down of Tgf-beta receptor I, Smad2 or Smad3 impaired ArflacZ induction by Tgf-beta2. Further, Tgf-beta2 failed to induce p19Arf in Cre-treated Tgfbr2 flox/flox MEFs, in which Tgf-beta receptor II was deleted. Interestingly, the p38 MAPK inhibitor SB203580 partially impeded Arf induction, showing cross-talk with Smad signals. Chromatin immunoprecipitation revealed that Tgf-beta2 induced the binding of Smad2/3 and RNA Polymerase II to genomic DNA proximal to Arf exon 1 beta. Increased promoter binding preceded the progressively induced Arf mRNA and primary Arf transcripts at 24 and 48 hours, indicating that the induction was due to increased transcription. In summary, our results indicate that Tgf-beta2 induces the Arf promoter via a direct Smad-dependent signaling pathway. This study provides the first evidence that Arf expression is coupled to cell extrinsic signals. We are currently exploring this pathway in other type of cells and testing whether Arf induction by oncogenes also depends on Tgf-beta signaling.

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 1097.
Abstract 11: Asymmetric division via nonrandom chromosomal cosegregation potentially defines a novel class of universal cancer stem cells
Authors: Xin Hong-Wu , Hari Danielle M. , Jaiswal Kshama R. , Kim Bo-Kyu , Ray Satyajit , Koizumi Tomotake , Wiegand Gordon W. , Steinberg Seth M. , Garfield Susan H. , Andersen Jesper B. , Factor Valentina M. , Thorgeirsson Snorri S. , Avital Itzhak
Published: February 2011

Cancer stem cells (CSC) are thought to be responsible for cancer initiation, maintenance, and therapeutic failure. A hallmark of stem cells is self-renewal via asymmetric cell division (ACD) into daughter stem cells and cells predestined for differentiation. In addition, recent reports questioned the ability of the gold standard for testing CSC (xenotransplantation into immunodeficient mice) to truly detect and test CSC. The aim of this study was to demonstrate fundamental stem cell's traits such as ACD with non-random-chromosomal cosegregation (ACD-NRCC) in cancers.

Here, we show that a unique self-renewal mechanism i.e. ACD-NRCC occurs in various human cancers using DNA double labeling and confocal microscopy. ACD-NRCC was found exclusively in a subpopulation of CSC (side population and CD133+ cells) and not in the non-cancer-stem-cells fractions. Cells that demonstrated ACD-NRCC also exhibited superior tumor initiation capacity in nude mice (p=0.028). Furthermore, we found that the niche provided by the non-stem cancer cells directly regulates self-renewal via ACD-NRCC. This regulation is dependent on a potentially novel heat sensitive soluble factor (Self Renewal Factor, SRF). Gene expression microarray data showed down-regulation of genes associated with ACD in tested cancers.

Detection of ACD-NRCC in various human cancers provides direct evidence for the existence of cancer cells with unique stem cells traits. This is the first report to demonstrate the fundamental stem cells trait of ACD-NRCC in human cancer. Detection of cells capable of undergoing ACD-NRCC in various cancers potentially defines a novel class of CSC with superior tumor initiating capacity. Using ACD-NRCC, we propose a universal method for testing, isolating and targeting CSC. The gene expression microarray data suggested that faulty mechanisms of ACD-NRCC might result in stem cells derived cancers. Further purification of the niche's self-renewal-signals and utilizing our methodology for isolation of CSC have far reaching implications in regenerative medicine, cancer genetics and potential novel cancer therapeutics targeting CSC via ACD-NRCC.

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 11.
Abstract 1102: Regulation of protein citrullination through p53/PADI4 network in DNA damage response
Authors: Matsuda Koichi , Tanikawa Chizu , Nakamura Yusuke
Published: February 2011

Upon wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However its involvement in post-translational modifications of proteins has not been well characterized so far. We here report the novel role of p53 in the regulation of protein citrullination. Citrullination (also referred as deimination) is one form of PTMs converting an arginine residue in protein to a citrulline residue. This reaction is mediated by a Ca2+-dependent enzyme called as peptidylarginine deiminase. Citrullination of proteins results in a loss of positive charge and causes a significant biochemical change. On the other hand, citrullinated peptides/proteins are recognized as a non-self protein and subsequently activate immune systems. Hence, protein citrullination is now considered to play a pivotal role in various human diseases, but any roles of citrullination in carcinogenesis were not reported so far.

We found p53 transactivated peptidylarginine deiminase type 4 (PADI4) through intronic p53-binding site and ectopic expression of p53 or PADI4 induced protein citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their citrullination, indicating the regulation of protein citrullination in a p53/PADI4 dependent manner. We identified that PADI4 citrullinated histone chaperon protein, nucleophosmin (NPM1) at arginine 197 residue in vivo under physiological condition. NPM1 was shown to continuously shuttle between the nucleus and cytoplasm and translocate from nucleoli to nucleoplasm as a response to DNA damage. Therefore, we investigated the effect of protein citrullination on subcellular localization of NPM1. Immunocytochemical analysis revealed that citrullination of NPM1 by PADI4 resulted in its translocation from nucleoli to nucleoplasm, while PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly knockdown of PADI4 attenuated p53-mediated growth inhibitory activity, demonstrating the significance of PADI4-mediated protein citrullination in the p53 signaling pathway.

Most of previous studies assessing enzymatic activities of PADIs were conducted under non-physiological condition using Ca ionophore or at higher Ca concentration. To our knowledge, this is the first report demonstrating the physiological regulatory mechanism of protein citrullination. Thus, p53 that can directly or indirectly modulate biological property of various proteins through the protein citrullination.

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 1102.
Abstract 1104: Perturbation of 60S ribosomal biogenesis results in ribosomal protein L5 and L11-dependent p53 activation
Authors: Sun Xiao-Xin , Dai Mushui
Published: February 2011

Ribosomal proteins play an important role in p53 activation in response to ribosomal stress. Multiple ribosomal proteins, including L5, L11, L23, and S7, have been shown to bind and inhibit MDM2, leading to p53 activation. However, it is not known whether ribosomal protein inhibition of MDM2 is specific to certain, but not all, ribosomal proteins. Here, we show that L29 and L30, two ribosomal proteins from the large ribosome subunit, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60S ribosomal biogenesis by knocking down either L29 or L30 drastically induced the level and activity of p53, leading to p53-depedent cell cycle arrest. Furthermore, this p53 activation requires ribosomal proteins L5 and L11, as knocking down either L5 or L11 completely abolished p53 activation induced by knockdown of L29 or L30. Consistently, knockdown of L29 or L30 enhances the interaction of MDM2 with L5 and L11. These results suggest that direct perturbation of the 60S ribosomal biogenesis activates p53 via L5 and L11-mediated MDM2 suppression.

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 1104.
Abstract 1115: Epigenetic regulation of BRCA-1 by xenobiotic and dietary ligands of the aryl hydrocarbon receptor
Authors: Romagnolo Donato F. , Selmin Ornella I. , Papoutsis Andreas J.
Published: February 2011

Mutations of BRCA-1 account for only 5-10% of breast cancer cases, whereas sporadic breast tumors represent the remaining 90-95%. In sporadic breast cancers there is silencing of BRCA-1 in the absence of mutations. Factors contributing to this etiology may include environmental ligands of the aryl hydrocarbon receptor (AhR) such as dioxins (ex.2,3,7,8 tetrachlorodibenzene(p)dioxin, TCDD) and polycyclic aryl hydrocarbons (PAHs). Dietary ligands of the AhR with potential protective effects include the phytoalexin resveratrol found in grapes. In this study, we investigated the mechanisms responsible for repression of BRCA-1 expression by the activated AhR. The results of DNA binding studies in MCF-7 breast cancer cells documented that TCDD-induced the association of the AhR to xenobiotic responsive elements (XRE) harbored in the BRCA-1 promoter region. The cotreatment with resveratrol reduced AhR recruitment in a dose-dependent fashion, prevented the TCDD-dependent repression of BRCA-1 transcription and restored BRCA-1 protein expression. The treatment with TCDD induced the recruitment of histone deacetylase-1, DNA methyltransferase-1, methyl-binding protein-2, and reduced the association of acetylated histone-4 and acetylated histone-K9. These effects were abrogated by cotreatment with resveratrol. We propose that dietary antagonists of the AhR such as resveratrol may be useful in the prevention of epigenetic BRCA-1 silencing.

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 1115.
Abstract 1128: The tumor suppressive function of Merlin/Nf2 is mediated by a novel tight junction-associated protein complex
Authors: Yi Chunling , Kissil Joseph
Published: February 2011

Mutations in the NF2 tumor suppressor gene lead to inherited cancer-disorder Neurofibromatosis Type 2 (NF2) and other sporadic nerve-sheath tumors. Merlin, the protein product of the NF2 gene, suppresses cell proliferation and tumorigenesis by regulating multiple signaling pathways including the Ras-MAPK pathway. Through affinity purification, we have identified a novel tight-junction-associated protein complex comprising Merlin and a number of tight junction associated proteins. We show that Merlin directly interacts and co-localizes with one of these tight junction proteins through their mutual coiled-coil domains in epithelial and Schwann cells. Furthermore, the two proteins co-localize to paranodes and Schmidt-Lantermann incisures in myelinated peripheral nerve, the most frequent site of tumor development caused by Nf2 inactivation. We establish that the novel Merlin-binding protein functions downstream of Merlin as a positive regulator of Rac1 activity and the Ras-MAPK signaling. Importantly, we are able to demonstrate that depletion of this Merlin-interacting protein in Nf2-/- schwannoma cells down-regulates the Ras-MAPK signaling pathway, and impedes cellular proliferation in vitro and tumor development in vivo. Taken together with our observations that several NF2 patient-derived mutant Merlin proteins exhibit diminished association with this protein and that its level is elevated in 9 out of 10 human schwannoma samples examined, we conclude that the novel Merlin-binding partner identified in this study mediates the tumor suppressive function of Merlin and represents a potential therapeutic target for NF2 related tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1128.
Abstract 113: The TCGA proneural subtype predicts improved clinical outcome for low-grade oligodendrogliomas
Authors: Moreno Carlos S. , Cooper Lee , Cholleti Sharath R. , Gutman David , Kurk Tahsin , Brat Daniel J. , Saltz Joel H.
Published: February 2011

The Cancer Genome Atlas Project (TCGA) has produced a wide array of omic data on glioblastoma multiforme (GBM) tumors, and has resulted in several works on the molecular subtyping of GBM tumors. Despite its richness, the TCGA dataset currently contains very limited data for low-grade gliomas and progressive GBMs, preventing analysis of progression models and prognostic biomarker discovery for these tumor types. A complementary dataset, the NCI Repository for Molecular Brain Neoplasia Data (REMBRANDT), includes a collection of samples for low-grade and progressive gliomas. This data contains some paired tumors that progressed to GBM and like TCGA, includes pathological and survival information. Our in silico study of low-grade and progressive gliomas has leveraged this complementary data to examine the progression of low-grade gliomas to GBM. The REMBRANDT dataset includes 176 samples with survival outcome data, including 101 grade IV GBM, 32 astrocytomas, and 43 oligodendrogliomas. Of the astrocytomas and oligodendrogliomas, there are 31 grade II samples and 31 grade III samples, with the remainder lacking grade data. We have investigated the gene expression profiles of the REMBRANDT samples to compare them to the established classes from TCGA and other studies. Candidate prognostic biomarkers have been identified to predict the progression of low-grade gliomas. We have found that the TCGA Proneural subtype predicts improved clinical outcome for low-grade oligodendrogliomas, with significantly better survival among all 176 low and high-grade samples (log rank test p = 1.27e-08). Moreover, analysis of 62 grade II and grade III samples determined that the Proneural class still has significantly better outcome (p= 0.0033). Further sub-analysis to control for oligodendrogliomas and grade, found that grade III oligodendrogliomas with the Proneural signature still had significantly improved survival (p < 0.05, n = 12). Stratification using the signatures developed by Li et al that define six oligodendrioma and glioma subtypes demonstrated that the OA subclass had significantly better survival (log rank test p = 9.14e-08). However, these signatures were not significant in multivariate analysis after controlling for grade and disease subtype. Thus, the gene expression patterns identified in the TCGA analysis of GMB has intrinsic prognostic value for low-grade oligondendriomas, and likely represents important differences in tumor biology with implications for treatment and therapy.

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 113.