bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1946: Silencing of EVI1 with siRNA inhibits the growth of ovarian tumor cells
Authors: Chang Bey-Dih , Heidel Jeremy D. , Primiano Thomas
Published: August 2012

There is currently no effective treatment for ovarian cancer. EVI1 (ecotropic viral integration site 1) is an oncogenic transcription factor that enables proliferation and inhibits apoptosis of ovarian tumor cells. Small interfering RNA (siRNA) drugs provide ideal means for perturbing intracellular oncogenic targets. Five (5) siRNA (21+21 nt) sequences were identified by in silico analysis that target the coding region of human EVI1 (siEVI1 A-E). Each of these siEVI1 species were transfected with Dharmafect4 at 25nM into SKOV3, ES-2, OVCAR3, OV-90, TOV-112D, PA-1, UWB1.289, and Coav-3 ovarian tumor cells. The non-tumorigenic cell line Hs832.Tc was used as a control. Four species (siEVI1 B-E) decreased the viability of each tumor cell line by at least 70% with none of the five siEVI1 species altered the viability of control cells, demonstrating the anti-proliferative effect of targeting EVI1 with siRNA. Both siEVI1-B and -D potently decreased the viability of ovarian tumor cells, having IC50 values of 0.8 and 0.2 nM, respectively, which compare favorably to an IC50 of 15 nM for paclitaxel. RT-PCR and immunoblot results showing dose-dependent decreases in EVI1 mRNA and protein using either siEVI1-B or-D are consistent with the mechanism of the anti-proliferative effect occurring via RNA interference. Further, dose-dependent increases in annexin staining upon siRNA treatment indicate that induction of apoptosis contributes to the decrease in cell viability, a result consistent with inhibition of EVI1 activity. These results suggest that EVI1 is a good target for ovarian cancer therapy and silencing by siRNAs is an effective means of triggering the death of ovarian tumor cells. These findings also suggest that anti-EVI1 siRNAs have the potential to be effective therapeutics for the treatment of patients with ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1946. doi:1538-7445.AM2012-1946
Abstract 1967: Multi-stage silicon vector-delivered tumor antigen and TLR ligands for dendritic cell-based cancer immunotherapy
Authors: Xia Xiaojun , Ferrari Mauro , Shen Haifa
Published: August 2012

Immunotherapy has shown promise in cancer treatment but achieved limited success in clinic trials. With this strategy, eliciting strong antitumor immune response in vivo remains a challenge, largely due to the immunosuppressive tumor microenvironment and inefficient delivery of cancer vaccine. Nanoparticle-delivered antigen together with adjuvants provides a promising strategy to increase the vaccine delivery efficiency. Recently, our group developed a multi-stage silicon vector (MSV) system for protected and prolonged nano-scale drug delivery. MSV delivered siRNA in vivo induced sustained knockdown of target gene expression and subsequent anti-tumor efficacy in mice. Here we tested the effect of MSV-formulated antigen peptides and Toll-like receptor (TLR) ligands on dendritic cells. By formulating HER-2 antigen peptide together with TLR4 ligand (MPLA) and TLR9 ligand (CpG), we found that MSV-delivered antigen and TLR ligands were uptaken by mouse dendritic cells after 1h co-incubation, and started to be released inside the cells at 3 h after co-incubation. Moreover, MSV-delivered TLR ligands strongly activated mouse dendritic cell expression of co-stimulatory molecules such as CD80 and CD86. It also induced dendritic cells to produce large amount of pro-inflammatory cytokines, such as TNF-alpha, IL-6, and IL-12. In summary, MSV-delivered vaccine efficiently delivered antigen and immuno-activating TLR ligands together into dendritic cells, and promoted dendritic cell maturation and activation. Therefore, MSV formulation of tumor antigen and TLR ligands may serve as a nano-scale cancer vaccine with enhanced in vivo efficacy. Ongoing experiments include testing in vivo T cell responses to MSV-delivered HER-2 antigen peptide and TLR ligands, and determining the antitumor efficacy on mouse mammary gland tumor model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1967. doi:1538-7445.AM2012-1967
Abstract 1976: Monocytic/macrophagic differentiation status of human promyelocytic leukemia cells dictates targeting efficacy of NF-kappaB downstream gene productes
Authors: Vézina Amélie , Annabi Borhane
Published: August 2012

Disruption of the endothelial blood-brain barrier (BBB) facilitates the translocation of circulating microbes and immune cells. As a consequence, secondary cerebral infections and development of brain pathologies may occur. Enhanced transendothelial migration of HL-60-derived macrophages was recently demonstrated, but the intrinsic signalling mechanisms involved poorly documented. In this study, we investigated NF-kB signalling pathway targets associated with phorbol 12-myristate 13-acetate (PMA)-induced promyelocytic HL-60 differentiation into macrophages. Given that the inhibition of matrix metalloproteinase (MMP)-9 and of cyclooxygenase (COX)-2 limits BBB disruption, we also investigated the efficacy of the chemopreventive and dietary-derived epigallocatechin-3-gallate (EGCG) molecule on the NF-kB-mediated transcriptional regulation of a panel of 89 biomarkers including MMP-9 and COX-2. PMA-induced HL-60 cell differentiation and adhesion were reflected by IkB degradation, a signalling process that was reversible by EGCG. Using gene expression arrays, we identified moderately-to-highly expressed NF-kB gene targets associated with HL-60 differentiation process, among which EGCG was able to efficiently inhibit gene and protein MMP-9 and COX-2 expression. In contrast, EGCG was not only inefficient in reversing some PMA-induced NF-kB genes in terminally-differentiated macrophages but rather showed selective antagonism towards COX-2 expression while that of MMP-9 still remained high. Decreased expression of the 67 kDa Laminin Receptor in terminally-differentiated macrophages may contribute to such differential EGCG efficacy. Our results suggest that MMP-9-mediated BBB disruption may be prevented by EGCG only during procarcinogenic processes that trigger HL-60 macrophagic differentiation. Terminally-differentiated macrophage transendothelial migration associated to neuroinflammation may however not be pharmacologically affected by such a specific class of chemopreventive flavonoid.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1976. doi:1538-7445.AM2012-1976
Abstract 1994: Honokiol reduces drug resistance by down-regulation of survivin expression in multidrug resistant squamous cell carcinoma of the head and neck
Authors: Wang Xu , Beitler Jonathan J. , Hong Hong Wang , Lee Michael J. , Chen Zhengjia , Koenig Lydia , Amin A. R. Ruhul , Chen Zhuo (Georgia) , Arbiser Jack L. , Shin Dong M.
Published: August 2012

Background: Resistance to chemotherapy is a major obstacle in cancer therapy. The purpose of this study is to evaluate honokiol in combination with paclitaxel in controlling proliferation of multi-drug resistant (MDR) cancer cells. Honokiol is a small molecule purified from Magnolia officinalis. Honokiol has been shown to suppress tumor growth through apoptosis and inhibition of angiogenesis. In this study, we investigated the effects and mechanisms of those effects of honokiol on MDR squamous cell cancers of the head and neck (SCCHN) Methods: Serial MDR SCCHN lines including KB-8-5, KB-C1, and KB-V1 derived from the drug sensitive parental KB-3-1 cells were used in this study. Cytotoxic effects of honokiol alone and in combination with paclitaxel on apoptosis in drug-sensitive and -resistant cells were evaluated in vitro and in a subcutaneous KB-8-5 xenograft model, Results: The cell growth inhibition analysis revealed a wide range of IC50 values of paclitaxel from 1.66±0.09 to 6560.9 ±439.52 ng/ml in the KB serial cell lines, indicating that those cell lines have different levels of resistance to the paclitaxel treatment. In contrast, honokiol effectively inhibited proliferation and induced apoptosis in all four cell lines with IC50 values from 3.35±0.13 to 2.77±0.22 μg/ml. Mechanistic studies revealed that honokiol induced similar mitochondria-dependent apoptosis in drug resistant cell lines regardless of the differential resistance levels to paclitaxel treatment. Moreover these effects were associated with inhibition of STAT3 phosphorylation and down-regulation of STAT3 target gene expression, including survivin and Mcl-1. Combined treatment with honokiol and paclitaxel synergistically increased cytotoxicity as compared to the single drug treatment. This combination also significantly inhibited growth of xenografted tumors in nude mice. Conclusion: These results suggest that honokiol may be a promising drug in overcoming paclitaxel resistence of SCCHN. Targeting MDR SCCHN using honokiol in combination with paclitaxel, may benefit patients with SCCHN. (Supported by grants P50CA128613, GCC Distinguished Cancer Scholar to Dong M. Shin and Jonathan J Beitler)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1994. doi:1538-7445.AM2012-1994
Abstract 2009: PKR induces apoptosis via PP2A activation and Bcl-2 dephosphorylation in IL-3 dependent murine bone marrow stem/progenitor cells
Authors: Cheng Xiaodong , Bennett Richard L. , Liu Xiangfei , May Stratford W.
Published: August 2012

The Interferon-induced double-stranded RNA-activated protein kinase (PKR) was originally found to be activated by viral infection leading to inhibition of new protein synthesis and apoptosis. Recently multiple studies have reported decreased expression and function of PKR in leukemia cells, although the exact role of PKR in the process of oncogenesis remains unclear. Since leukemias are clonal disorders originating in a primitive pluripotent leukemic stem cell (LSC) which is regulated by multi-potential hematopoietic growth factors including interleukin-3 (IL-3). Thus, studying how PKR regulates apoptosis in IL-3 dependent (CD123+) pluripotent stem/progenitor cells is expected to reveal PKR's role in leukemia. PKR expression was either knocked down (by 78%) using a specific ShRNA or directly inhibited (57%) by treating cells with a specific PKR inhibitor. Results revealed that inhibition of PKR was associated with a reduction of PP2A activity, a 2.3 fold increase in Bcl-2 phosphorylation and inhibition of apoptosis following treatment with 100uM hydrogen peroxide for 48 hours. In addition, okadaic acid (an inhibitor of PP2A) had a similar effect to PKR inhibition on both Bcl-2 phosphorylation (1.74 fold increase) and stress-induced apoptosis. By contrast, treatment of cells with the PP2A activator, FTY 720, resulted in decreased Bcl-2 phosphorylation (by 2.24 fold) and enhanced apoptosis in response to treatment of cells with chemotherapy (etoposide or doxorubicin) or hydrogen peroxide. To test the role of PKR in growth factor- withdrawal mediated apoptosis in bone marrow stem/progenitor cells, CD123+(IL-3R[[Unsupported Character - Symbol Font ]]+) bone marrow cells from wild type, PKR transgenic (over-expressed) and PKR null(-/-) mice were exposed to different stresses including IL-3 deprivation or hydrogen peroxide. CD123+ cells isolated from PKR null mice had a significantly lower percentage of apoptosis while CD123+ cells from PKR transgenic mice had increased apoptosis compared to cells from wild type littermate mice. These results demonstrate that PKR negatively affects hematopoietic cell survival by enhancing apoptosis following stress in a mechanism that involves PP2A activation and Bcl2 dephosphorylation. Taken together these results may indicate that inhibition of PKR's pro-apoptotic function in bone marrow pluripotent stem/progenitor cells may contribute to tumorigenesis in hematopoietic malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2009. doi:1538-7445.AM2012-2009
Abstract 2019: Investigation of cell death mediated by poly(ADP-ribose), Apoptosis-Inducing Factor (AIF) and caspases in breast cancer cells by RNA interference
Authors: Feng Xiaoxing , Proctor Alicia , Hopkins Mandi , Liu Mengwei , Koh David W.
Published: August 2012

Apoptosis-Inducing Factor (AIF) was previously characterized as a poly(ADP-ribose) (PAR) dependent, caspase independent cell death effector in a wide spectrum of diseases or conditions that involve cell death. The objective of this study was to determine the role of PAR and AIF in cancer cell death after chemotherapy. We have previously demonstrated the presence of AIF mediated caspase-independent cell death in MDA-MB-231 breast cancer cells following treatment with the DNA alkylating agent N-methyl-N’-nitrosoguanidine (MNNG). To gain further insight into the role of AIF in cell death induced by chemotherapy, we utilized RNA interference (RNAi) to knockdown expression of AIF and PAR glycohydrolase (PARG) in MDA-MB-231 and MCF-7 human breast cancer cells. UV-C irradiated AIF-silenced cells displayed increased cell viability. Moreover FACS analysis showed that AIF knockdown successfully protected cells against apoptosis induced by MNNG and the chemotherapeutic agents epirubicin and cyclophosphamide. Interestingly, the knockdown of PARG led to decreased apoptotic cell death after treatment with MNNG. However, inhibition of caspases using Q-VD-OPh in PARG RNAi treated cells led to an increase in apoptotic cell death, which suggests that caspase dependent cell death after chemotherapy is mediated in part by PARG. However, the knockdown of AIF in these cells led to significant protection, which indicates that cell death in the absence of caspase activity in PARG knockdown cells is mediated by AIF. Taken together, the results provide a paradigm where AIF can substitute as caspase executioner in the absence of PARG and caspases in breast cancer cells. Thus, the activation of AIF mediated cell death is a promising strategy to improve the treatment of breast cancer by providing an alternative pathway for tumor eradiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2019. doi:1538-7445.AM2012-2019
Abstract 2022: P53-dependent thrombocytopenia induced by the histone deacetylase inhibitor S78454
Authors: Ali Ashfaq , Bluteau Olivier , Palazzo Alberta , Boukour Siham , Lordier Larissa , Rameau Philippe , Leceluse Yann , Kraus-Berthier Laurence , Jacquet-Bescond Anne , Lelièvre Hélène , Depil Stéphane , Solary Eric , Raslova Hana , Vainchenker William , Plo Isabelle , DEBILI Najet
Published: August 2012

S78454 (also called PCI-24781) is an orally bioavailable, hydroxamate-based pan-HDACi currently being tested in clinical trials in the US and EU. Like many other HDACi, this drug induces a reversible thrombocytopenia. This thrombocytopenia, which was associated with a decrease in either GATA1 transcriptional activity or the expression of proteins of the Rho GTPase family (RhoA, Cdc42 or Rac), remains poorly understood. Given that the S78454 plasma level is above 100nM in treated patients, we performed a dose-response analysis (from 10 to 100 nM) of the drug effects on CFU-MK growth and cell proliferation. CFU-MKs were generated by ex vivo culture of CD34-positive cells and their differentiation was dose-dependently decreased after either a 24-hour treatment or a continuous exposure to S78454. This effect was associated with a dose-dependent decrease in MK proliferation. When added at day 8 of the culture, at a time-point when MK have nearly finished their endomitotic process and are entering in the cytoplasmic maturation step, S78454 induced a dose-dependent decrease (ten- to- two fold at 100nM and 20nM, respectively) in proplatelet formation, even when secondarily removed from the culture medium. The decreased proliferation was associated with an increased apoptosis, as demonstrated by the presence of a sub-G1 peak after propidium iodide staining, Annexin V binding, and caspase-3 proteolysis. We did not observed any blockade in the TPO/MPL/JAK2 signaling since ERK, Stat3 and Stat5 phosphorylation remained unaffected. Interestingly, MK exposed to S78454 displayed an increase in γH2AX foci formation and a decrease in Rad51 expression, a major mediator of homologous recombination, indicating a defective repair of DNA double-strand breaks. Consequently, P53 became phosphorylated in MKs, and the expression of its target genes BAX and P21 was increased. Altogether, these results suggest that S78454-induced thrombocytopenia may be mediated by induction of apoptosis in MK due to DNA-double-strand breaks and p53 activation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2022. doi:1538-7445.AM2012-2022
Abstract 2027: Carbamylated albumin increase MMP9 activity via ERK MAPK pathway
Authors: Kim Jin Young , Park Keon Uk , Shin So Jin , Hwang Il Seon , Ha Eunyoung , Ryoo Hun-Mo
Published: August 2012

Objective: Carbamylation, the binding of cyanate to amino acid lysine residues, is a post-translational modification. Pathophysiological consequences of carbamylation and adverse effects of carbamylated proteins remain poorly understood. This study was designed to influence of carbamylated albumin on matrix metalloproteinase(MMP) expressions in rat mesangial cells (RMC). Methods and Results: The effect of carbamylated albumin was evaluated using zymography, Western blot analysis, MMP-9 promoter luciferase assay, and ELISA. We found that carbamylated albumin significantly increased MMP-9 productions in RMCs via stimulating MMP-9 promoter activity and ERK1/2 MAPK phosphorylation. DecreasedTIMP-1 activity by carbamylated albumin was not observed. Conclusion: We demonstrated that carbamylated albumin increased MMP-9 activity via ERK1/2MAPK mediated pathway and further studies as to the functions of carbamylated albumin will be of merit.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2027. doi:1538-7445.AM2012-2027
Abstract 2028: Krüppel-like factor 4 null mouse embryonic fibroblasts exhibit DNA repair defects post exposure to gamma-irradiation
Authors: Engda Hagos , Yang Vincent W. , Yu Bing
Published: August 2012

BACKGROUND: Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that exerts a potent inhibitory effect on cell cycle progression. KLF4 functions as a tumor suppressor in colorectal cancer and is involved in regulating centrosome duplication. Abnormal amplification of centrosomes has been shown to contribute to genetic instability by increasing the frequency of mitotic defects. AIM: To determine whether KLF4 is involved in maintaining genetic stability during cell division. METHODS: Mice heterozygous for the Klf4 alleles (Klf4+/−) on a C57BL/6 background were crossbred. Mouse embryonic fibroblasts (MEFs) that are wild type (Klf4+/+), heterozygous (Klf4+/−) or null (Klf4−/−) for the Klf4 alleles were derived from day-13.5 embryos. Post-senescence MEFs were derived by passing cells to senescence and maintaining them in culture until they spontaneously regained proliferative. Immuno-detection of histone variant γ-H2AX was performed to assess chromosome integrity. The levels of cell cycle regulators such as Cdk2, Cyclin E, and Cyclin D were determined by Western blotting. The presence of double strand breaks (DSB) was determined by western blot and immunofluorescence for histone variant -H2AX contains nuclear foci. Flow cytometry has been used to determine the level of DNA damage in both wildtype and klf4 null cells following gamma-irradiation. Furthermore, Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. RESULTS: Cells that exhibits DSBs in their DNA can develop genomic instability, which can result in cancer formation. Compared to Klf4+/+ and Klf4−/− MEFs exposed to α-irradiation showed time-dependent increase in levels of histone variant -H2AX that persisted for up to 48 hours after the α-irradiation. On the other hand, Klf4+/+ halt cell cycle progression to allow repair of DNA damage. Western blot analysis showed that Cdk2, Cyclin E, Cyclin D and histone variant -H2AX expression were high in Klf4−/− MEFs. Furthermore, we found many of the down-regulated genes in Klf4-null MEFs encode DNA repair, and antioxidant genes. CONCLUSIONS: Results of this study demonstrate that Klf4 null mouse embryonic fibroblasts exhibit an impaired DNA damage response post gamma irradiation, when compared to wild-type cells. This data strongly suggest that the role for Klf4 in DNA damage repair in mouse embryonic fibroblast cells. These results indicate that KLF4 plays a crucial role in maintaining genetic stability and support the previous findings that KLF4 is a tumor suppressor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2028. doi:1538-7445.AM2012-2028
Abstract 2032: A novel function of WW domain binding protein 2 (WBP2) in regulating cytoskeletal function and cellular division through binding to co-chaperone BAG3
Authors: McCollum Andrea K. , Angelos Mathew G. , Fischione Andrea D. , Mineo Marco , Kohn Elise C.
Published: August 2012

BAG3, a BAG co-chaperone family member, co-localizes with actin microfilaments and influences processes that involve actin function, including adhesion and migration. Because actin function regulates cell division, we hypothesized that the BAG3 WW-domain regulates cellular division through its cytoskeletal association. We show that BAG3 co-localizes with leading lamellipodia exclusively during G2 and mitosis phases. Co-immunoprecipitation of BAG3 and pulldown of GST-tagged BAG3 WW-domain confirmed that BAG3 WW-domain association with actin increased during G2 and mitosis relative to asynchronous cells. Deletion of the BAG3 WW-domain caused an abnormal cellular morphology, degenerate F-actin organization, longer doubling time (p<0.05, n=4), and three-fold increase in polynucleated cells relative to empty vector and full length-BAG3 (FL-BAG3; p<0.001, n=3). We confirmed polyploidy development and absence of cytokinesis in αWW-BAG3 cells by live-cell imaging, and observed mitotic delay relative to control and FL-BAG3 cells. To identify a potential regulation mechanism for BAG3-actin binding, we identified WBP2 as a novel BAG3 WW domain interacting protein in a yeast two-hybrid assay. BAG3 and WBP2 co-immunoprecipitated only in G1 and S phases, indicating cell cycle-specific BAG3-WBP2 binding. WBP2 silencing caused indiscriminate BAG3 co-immunoprecipitation with actin across all cell cycle phases, suggesting that association with WBP2 prevents BAG3-actin binding. We are the first to identify a function for WBP2, which our data show regulates BAG3 WW-domain association with actin in a cell cycle-specific manner. Because actin dysfunction has been proposed to contribute to chromosomal instability and tumor formation, the WW-domain of BAG3 is a potential target to reduce abnormal cell division.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2032. doi:1538-7445.AM2012-2032
Abstract 2048: Phosphorylation and regulation of PTEN by Polo-like kinase 1
Authors: Choi Byeong Hyeok , Lee Kyung S. , Dai Wei
Published: August 2012

The tumor suppressor PTEN plays a critical role in the regulation of cell survival, proliferation, and angiogenisis. Deregulated expression and/or activities frequently result in tumor development. Several recent studies have revealed that PTEN also localizes to the nucleus and that nuclear PTEN may regulate the level and activity of mitotic checkpoint proteins, thus suppressing chromosomal instability during cell division. However, the mechanism by which PTEN is translocated to the nucleus remains unclear. Given that PTEN is heavily phosphorylated and that Plk1 is an important mitotic kinase, we have asked whether Plk1 directly phosphorylates PTEN and whether Plk1-mediated phosphorylation of PTEN functions as an important regulatory mechanism during cell division. Here we report that a significant fraction of PTEN localized to nuclear chromatin. The chromatin association of PTEN was enhanced during mitosis, which was correlated with an increase in PTEN phosphorylation at threonine 366 (T366) and serine 370 (S370). Using purified recombinant proteins, we demonsatrated that Plk1 was at least partly responsible for this phosphorylation. Plk1 strongly catalyzed the incorporation of 32P into PTEN, resulting in increased signals detected by a phospho-specific antibody that recognized T366/S370 phosphorylation. Furthermore, BI-2536, a potent Plk1 inhibitor, significantly blocked PTEN phosphorylation by Plk1 in vitro, as well as its chromatin association in vivo. Combined, these results strongly suggest that Plk1 phosphorylates PTEN at T366 and/or S370 and that this phosphorylation promotes its association with chromatin during the cell cycle.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2048. doi:1538-7445.AM2012-2048
Abstract 2049: Preclinical profile of AMG900 in combination with HDACIs in prostate cancer
Authors: Paller Channing , Wissing Michel , Kim Eugene , Hammers Hans , Rudin Charles , Carducci Michael , Kachhap Sushant
Published: August 2012

Background: Histone deacetylase (HDAC) enzymes maintain the structural conformation of chromatin. Inhibition of HDACs induces G1 arrest and differentiation. Previous microarray analysis demonstrated downregulation of the aurora kinase-pathway in prostate cancer cell lines treated with HDAC-inhibitors (HDACIs) vorinostat and valproic acid (VPA). Hence we sought to combine these HDACIs with AMG900, a pan-Aurora kinase inhibitor, to assess potential additive or synergistic effects, hypothesizing that low-dose combinations will have a proapoptotic effect comparable to a higher dose of AMG900 as a single agent. Methods: We used MTS assays, clonogenic assays, beta-galactosidase staining and western blot analyses to investigate differences in proliferation and molecular markers with combinations of AMG900 and HDACIs compared to AMG900 alone. Results: In vitro treatment of a panel of prostate cancer cell lines with low-dose combinations of AMG900 with HDACIs revealed enhanced antiproliferative responses compared to single agent use. Short term MTS assays only demonstrated moderate synergism in DU-145 and LNCaP cells co-treated with VPA and synergism in PC3 cells co-treated with vorinostat, based on the Chou-Talalay-method (CalcuSyn). However, all cell lines demonstrated significant decreased long term survival when combining low-dose AMG900 (1nM) with HDACIs. In DU-145 and PC3 cells lines, the low-dose AMG900 (1nM) combinations demonstrated equivalent clonogenic survival to high dose AMG900 treatment (5nM), either alone or in combination with HDACIs. We noted morphologic changes in treated cell lines and explored senescence with beta-galactosidase staining. DU-145 and PC3 cells, when treated with a combination of AMG900 and HDACIs, demonstrated a significant increase in beta-galactosidase positive cells, suggesting cell senescence (10-20% in single agent vs 50% in low-dose combinations). We also explored the mechanisms associated with decreased clonogenic survival at low doses and found a significant increase in cleaved PARP in DU-145 cells with both combinations, and with Vorinostat in LNCaP cells, but not PC3 cells which have a baseline level of ongoing apoptosis. In addition, western blot analysis showed significant differences in ACH3, pH3 and Cyclin B1 in combination treatment compared to single agent use. Conclusions: A combination of low-dose AMG900 with HDACIs in prostate cancer cell lines demonstrates synergy and efficacy equivalent to high doses of AMG900 alone, confirming our hypothesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2049. doi:1538-7445.AM2012-2049
Abstract 2059: NADPH Oxidase 4 induced ROS regulates anoikis resistance through activation of Src and EGFR.
Authors: Kim Hyeryeong , Park Eun-Kyung , Koo Kyung Hee , Park Byung-Kiu , Kim Yong-Nyun
Published: August 2012

Since cancer cells must overcome the anoikis for the metastasis, anoikis resistance is an important process for tumor progression and metastasis. To identify anoikis resistance mechanism, we performed mRNA microarray analysis with attached and suspended human lung epithelial adenocarcinoma A549 cells. Our microarray data reveals that Nox4 mRNA is up-regulated in the suspended cells. We evaluated both mRNA and protein levels of Nox4 and found that Nox4 is up-regulated in both mRNA and protein levels. In addition, higher levels of ROS are detected and Src activation was enhanced in suspended cells than attached cells. Interestingly, increased level of ROS in suspended cells was decreased by ROS scavengers or Nox4 inhibitors, leading to inactivation of Src and EGFR. Furthermore, knock-down of Nox4 by si-RNA specific for Nox4 resulted in inactivation of Src and EGFR. Because Src activation is not inhibited by EGFR inhibitor, Src activation appears to be independent on EGFR. In addition, si-RNA for Nox4 transfection decreased viability of cells in suspension. These results suggest that upon cell detachment, Nox4 expression is up-regulated and thus ROS levels are increased, which are important for activation of Src and EGFR, leading to sustained cell viability in the suspended cells. Therefore, we postulate that ROS generation through the Nox4 is an important process for anoikis resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2059. doi:1538-7445.AM2012-2059
Abstract 206: A single nucleotide polymorphism (SNP) rs35301225 (A/C/T) in pre-miR-34a alters the cell cycle regulation
Authors: Srivastava Anvesha , Ramalinga Malathi , Suy Simeng , Collins Sean P. , Kumar Deepak
Published: August 2012

Prostate cancer (PCa) is the most common type of cancer in men in the United States and is expected to result in ∼218,000 new cases and about 32,000 deaths in 2010. Recently, microRNA miR-34a has been associated with many cancers including prostate cancer. It has been shown that tumor suppressor p53 transcriptionally regulates the expression of microRNA- miR-34a, which confers translational inhibition and mRNA degradation of genes involved in cell cycle control and apoptosis. Several GWAS studies have reported that Single nucleotide polymorphisms (SNPs) in microRNA coding genes (both in seed and mature sequence) or their target binding sites may alter the strength of miRNA binding and deregulate target gene expression. To our interest we aimed to explore the effect of germ line polymorphism rs35301225 (A/C/T) residing in miR-34a (rs35301225) on the cell cycle progression and apoptosis. Prostate (PC3 and LnCap) and breast cancer (MDA-MB 231) cell lines were transfected with pre miRNA mimics containing A/C/T alleles (rs35301225) of miR-34a and the expression of CYCLIN D1, CDK4, CDK6 and proteins associated with cellular signaling cascades, involved in cell cycle regulation were determined by Western blotting. Luciferase assay was performed for the target evaluation. The cell proliferation and apoptosis were measured by WST-1 assay and flow cytometry respectively. The expressions of apoptosis-related proteins were detected by Western blotting in the transfected cells. Western blot analysis showed that CYCLIN D1, CDK4, CDK6 and other downstream molecules were significantly down-regulated in the presence of C allele, whereas no such reduction of expression was observed in the presence of A or T allele. Cell proliferation assay showed that different alleles of miR-34a also exert profound effect on proliferation pattern of cells. Flow cytometry analysis also showed that cells with different alleles of miR-34a may exert significant effect on cell cycle progression and apoptosis. To explore if this phenomenon was confined to prostate cancer cell lines or different alleles of mir-34a may alter cell function in general, experiments were repeated with a breast cancer cell line, MDA-MB-231. Interestingly the results obtained in breast cancer cell lines were similar to that obtained in prostate cancer cell lines. The results suggest that genetic variants residing in the mature miRNA may exert a substantial effect on their expression and function, which needs to be explored along with the biological effect of miRNAs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 206. doi:1538-7445.AM2012-206
Abstract 2061: Implication of NQO1 knockdown on prostate carcinogenesis
Authors: Thapa Dinesh , Kumar Addanki P. , Ghosh Rita
Published: August 2012

NAD(P)H-Quinone Oxidoreductase 1 (NQO1) is a key antioxidant enzyme that protects against quinone derived reactive intermediates and maintains a cellular pool of antioxidants. Recent studies from our laboratory and others have been found that the expression levels of NQO1 and several GST family genes are significantly suppressed in prostate tumors in preclinical mouse models. In this study, we investigated the role of NQO1 in prostate carcinogenesis. First, we characterized basal NQO1 expression in normal prostate cells (RWPE-1), initiated prostatic epithelium (BPH-1) and prostate cancer cells (LNCaP, C42B, PC-3 and DU145). Unlike in hormone-induced tumors in vivo, in-vitro study showed increased NQO1 expression in cancer cells compared to normal cells, and that finding was consistent with other cancer cell lines such as bladder, pancreatic and melanoma cells. Stable RNAi-mediated knockdown of NQO1 in prostate cancer cells increased cell proliferation and susceptibility to oxidant treatment. Furthermore, whole genome microarray revealed that NQO1 knockdown in LNCaP cells resulted in 1603 signature genes being deregulated. COL12A1, MCP-1/CCL2, IL-8, IL1R2 mRNA were among those that showed most highly significant increase (>6 fold; p<0.01)., whereas mRNA for nucleoplasmin 2, phosphotyrosine domain interacting protein PID1 and the melanoma tumor specific antigen gene family member MAGEC1 were highly significantly decreased (>12 fold; p<0.01). Investigations are under way to determine whether these genes are direct or indirect targets of NQO1. Thus, antioxidant NQO1 plays a significant but paradoxical role acting as a double-edged sword to regulate cellular processes. However, the mechanism by which the differential expression of these genes is regulated through NQO1 still remains to be elucidated. Blocking NQO1 may regulate ARE-related transcription factors, which in turn, possibly control antioxidant and pro-inflammatory pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2061. doi:1538-7445.AM2012-2061
Abstract 2066: Identification of NRF2 target genes in human cells: Aldoketo reductases as a potential biomarker for NRF2 activity
Authors: Choi Bo-hyun , Jung Kyeong-Ah , Song Mingu , Kwak Mi-Kyoung
Published: August 2012

The transcription factor NF-E2-related factor 2 (NRF2) plays a critical role in cellular defense system by up-regulating multiple antioxidant genes. Therefore, the identification of biomarker genes reflecting NRF2 activity would be important for the prediction of high-risk populations to environmental stresses. In the current study, we have investigated human genes of which expression is highly dependent on NRF2 to establish biomarker genes for NRF2 activity. For this purpose, NRF2-specific interfering RNA was stably introduced in normal human renal epithelial cells (HK-2) and human bronchial epithelial cells (NL-20), and changes in inducible genes were determined following treatment with 4 types of NRF2 activators: thiol-reacting sulforaphane, free-radical generating tert-butylhydroquinone, Michael acceptor cinnamic aldehyde and pro-oxidant hydrogen peroxide. These treatments showed relatively common alterations in gene expression, and among these, the expression of aldoketo reductase (AKR) 1C1 was highly inducible by all of these treatments in an NRF2-depedent manner. The levels for AKRs were found to be constitutively high in renal carcinoma A498 cells, of which NRF2 activity is elevated compared to normal renal epithelial HK-2 cells. In addition, the expression of AKRs was greatly enhanced by NRF2 activator treatments in human monocytes (U937), implying the potential utility of AKRs as a peripheral NRF2 marker. In conclusion, our results indicate that the expression of AKRs is highly dependent on NRF2 in human cells; therefore AKRs can be an effective biomarker for predicting NRF2 activity in human cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2066. doi:1538-7445.AM2012-2066
Abstract 2067: Putative role of Nrf2 as a tumor suppressor through inhibition of angiogenesis
Authors: Funes Juan M. , Kaufman Rachel , Flanagan James M. , Henderson Stephen , Robson Mathew , Pedley Barbara , Moncada Salvador , Boshoff Chris
Published: August 2012

An increase in reactive oxygen species (ROS) is a common biochemical change in cancer cells. To investigate the mechanisms of ROS accumulation during the process of tumorigenesis we employed a well-characterized model of stepwise human mesenchymal stem cell (MSC) transformation. Transformed MSC (tMSC) became dependent upon oxidative stress, as treatment with antioxidants impaired their tumorigenic potential. The increased ROS in tMSC was a consequence of a striking global down-regulation of the cellular antioxidant machinery correlating with reduced levels of the transcription factor Nrf2, a key regulator of the cellular antioxidant response. Expression of constitutive active versions of oncogenic H-Ras, Raf and PI3K/AKT led to decreased levels of Nrf2 in non-transformed MSC. Down-regulation of Nrf2 plays a central role in the defective ROS-scavenging capacity and oncogenic potential of tMSC, since its over-expression induces antioxidants, reduces the levels of ROS and impairs in vivo tumor growth. Mechanistically we show that restoration of Nrf2 expression sensitizes transformed cells to apoptosis and reduces an angiogenic response. Elevated Nrf2, in turn, led to destabilization of HIF-1α and decreased expression of angiogenic factors such as adrenomedullin and VEGF during hypoxic conditions. Moreover, we found a significant reduction in both proliferation and sprouting of human umbilical vein endothelial cells after being cultured in hypoxic/conditioned media from tMSC over-expressing Nrf2 compared with control. Down-regulation of Nrf2 during human cell transformation is not restricted to MSC as Nrf2 expression was also down-regulated when human mammary epithelial cells (HMEC) were genetically modified and transformed with the same oncogenic elements used for MSC. We performed microarray comparison studies and observed that expression of Nrf2 was down-regulated in a panel of human tumors. Furthermore, expression of Nrf2 and its downstream gene NQO1 was enhanced in breast cancer cell lines such as MCF-7 and MDA-MB-231 when the cells were treated with U0126, an inhibitor of ERK1/2 pathway. These results suggest that down-regulation of Nrf2 expression through activation of Ras/Raf/ERK pathway is not unique to in vitro transformed MSC or HMEC, but also occurs in established human breast cancer cell lines. Overall, our results indicate that Nrf2 expression is reduced in certain human tumor cells by mechanisms that involve activation of Ras signaling, and reveal a new role for Nrf2 as a tumor suppressor through inhibition of angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2067. doi:1538-7445.AM2012-2067
Abstract 207: Casitas B-lineage lymphoma-3 is an epigenetically regulated gene whose knockdown induces mesenchymal to epithelial transition and enhances erlotinib sensitivity in lung cancer cell lines
Authors: Zhang Jing , Giri Uma , Cantu Krystha , Wang Jing , Beketaev Ilimbek , Byers Lauren , Saintigny Pierre , Heymach John V. , Lin Steven H.
Published: August 2012

Casitas B-lineage lymphoma (Cbl) proteins are E3 ubiquitin ligases and multifunctional adaptor proteins that are implicated in the regulation of signal transduction in various cell types and in response to different stimuli. Cbl-3 is a distinct member of this family of 3 genes that is specifically expressed in the epithelial lining of organs, particularly the bronchial airways, whose function, especially in cancer cells, is relatively unknown. Here we report that Cbl-3 expression and promoter CpG island DNA methylation differed profoundly in epithelial compared to mesenchymal-like lung cancer cells. 5-azacytidine treatment, as well as DNA methyltransferase-1 (DNMT1) KD (but not DNMT-3a or 3b) enhanced promoter demethylation and Cbl-3 gene expression. Knock down (KD) of Cbl-3 in mesenchymal-like lung cancer cell lines strongly enhanced EGFR expression and activated downstream pathways. As a result, cells that were normally resistant to erlotinib became greatly sensitized to drug treatment by Cbl-3 KD. Additionally, Cbl-3 KD inhibited cellular migration and increased cellular adhesion to the extra cellular matrix (ECM) as well as promoted cell-to-cell interaction through enhanced E-Cadherin and FAK expression. These alterations suggested a mesenchymal to epithelial transition (MET) conferred by Cbl-3 KD. Take together, this data provided new insights into Cbl-3 gene regulation and elucidated a potential role of Cbl-3 in EGFR signaling in the context of EMT in lung cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 207. doi:1538-7445.AM2012-207
Abstract 2072: Distinct antioxidant profiles of human prostate carcinoma cell lines and tissues: Implications for rational design of redox therapy
Authors: Chaiswing Luksana , Zhong Weixiong , Oberley Terry D.
Published: August 2012

Several cancer chemotherapeutic drugs and radiation mediate their effects, at least in part, by generation of reactive oxygen/nitrogen species. Therefore, the levels of antioxidants and status of cellular redox state have tremendous influence on the outcome of these therapies. We analyzed antioxidant proteins in normal prostate epithelial cells and tissues as well as prostate cancer cell lines and tissues. Highly aggressive androgen-independent prostate cancer PC3 and LNCAP-C42B cells demonstrated significantly higher levels of superoxide dismutase (SOD) 2, peroxyredoxin (Prx) 1, 2, 3, and 4, thioredoxin (Trx) 1 and 2, and thioredoxin reductase (TR) 1 and 2, relative to normal prostate epithelial PrEC cells and immortalized RWPE1 human prostate epithelial cells. Similarity, the highly aggressive human prostate cancer tissues (Gleason scores 4+4 and 4+5) demonstrated significantly higher levels of SOD1 and 2, Prx 3 and 4, Trx 1 and 2, and TR 1 and 2 relative to adjacent normal prostate tissues. In contrast, the expression levels of these antioxidant proteins were lower or were not altered in less aggressive human prostate cancer tissues (Gleason score 3+3) relative to adjacent normal prostate tissues. Utilizing Trx1 redox western as a marker of cellular redox states, we found more oxidized form of Trx1 in PC3 and LNCAP-C42B cells and human prostate cancer tissues (Gleason scores 4+4 and 4+5) despite their higher levels of Trx1 protein expression. Our data suggest that alteration in antioxidant proteins and cellular redox state may differentiate cancers from non-cancer tissues. Additionally, knowledge of redox state profiles can help to predict the response of each cancer cell type to chemotherapeutic drugs and radiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2072. doi:1538-7445.AM2012-2072