bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 47: Cancer-associated fibroblasts mediate extracellular matrix remodeling and three-dimensional invasion of scirrhous gastric carcinoma cells
Authors: Yamaguchi Hideki , Yanagihara Kazuyoshi , Yashiro Masakazu , Sakai Ryuichi
Published: August 2012

Scirrhous gastric carcinoma (SGC; also called diffusely infiltrative carcinoma, Borrmann's type-IV carcinoma, or the linitis plastica-type carcinoma) have the worst prognosis among various types of gastric cancer, owing to highly progressive invasion, frequent metastasis to lymph nodes, and high incidence of peritoneal dissemination. SGC cells rapidly and diffusively infiltrate through the submucosa without forming obvious tumor mass and induce extensive stromal fibrosis, resulting in thickening and stiffening of the gastric wall. Because histophathological analyses have shown that massive proliferation of stromal fibroblasts occur within lesions of SGC, cancer-associated fibroblasts (CAFs) have been proposed to support the progression of SGC. However, biological and molecular basis of the interaction between SGC cells and CAFs remains largely unknown. In this study, we investigated the roles of CAFs in invasion and extracellular matrix (ECM) remodeling by SGC cells. When 44As3 SGC cells were co-cultured with CaF37 SGC-derived fibroblasts on 3D Matrigel, they formed large cell aggregates that invade into the Matrigel. Other SGC cell lines, HSC59 and HSC44PE, but not differentiated non-SGC gastric caner cell lines, MKN7, MKN1, and MKN74, formed invasive cell aggregate with CaF37 cells. Time-lapse imaging of SGC and CaF37 cells, and of fluorescent microsphere embedded in the Matrigel revealed that this process is associated with extensive contraction and remodeling of ECM. In order to further characterize the ECM remodeling activity of CAFs, we cultured 44As3 and CaF37 cells on fluorescent gelatin-coated coverslips. CaF37 cells caused disruption of the gelatin matrix and this activity was further promoted by co-culturing them with 44As3 cells. Immunoblotting and immnofluorescence analyses revealed that phosphorylation of myosin light chain was significantly increased in CaF37 cells co-cultured with 44As3 cells. Finally, a myosin II inhibitor blebbistatin blocked the three-dimensional invasion and ECM remodeling by 44As3 and CaF37 cells. These results indicate that SGC cells promote actomyosin-mediated contractility of stromal fibroblasts to remodel ECM during invasion.

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 47. doi:1538-7445.AM2012-47
Abstract 470: Motility of human colorectal cancer cells is promoted by H1047R kinase mutation of PI3K p110-alpha
Authors: Wan Guanghua , Ongchin Melanie , Hand Essence , Rajput Ashwani
Published: August 2012

INTRODUCTION: Mutations in the catalytic subunit p110α of Class 1A phosphatidyinositol 3-kinase (PI3K) occur in nearly 1/3 human colorectal cancer (CRC). In vivo studies have demonstrated that cell lines bearing the p110α mutation are more metastatic than wild type PI3K cells. The most frequent cancer-specific mutations in p110α are the kinase domain mutation H1047R and the helical domain mutation E542K/E545K. Our group has previously shown that the two hotspot mutations in PI3K in either the helical or kinase domains have a significantly different metastatic potential in an orthotopic murine model. The aim of this study was to compare the difference in cell motility between the helical and kinase domain mutation bearing human CRC cell lines. METHODS: Wound healing Assay was used to determine the motility of human CRC cells in vitro. Effect of PI3K inhibition on cell motility was tested under the treatments with 0.1 and 1.0 µM of wortmannin, which is a class 1 PI3K inhibitor, and PIK75, which is the specific inhibitor of p110α. RESULTS: Our data indicate that H1047R-p110α increases cell motility more than E545K-p110α and the inhibition of PI3K significantly delayed or abrogated the migration of CRC cells. Immuno-blotting analysis showed increased downstream survival signaling pathway activation of PI3K in the cells bearing H1047R-p110α knase domain mutation compared to the cells which have E545K-p110α helical domain mutation. CONCLUSION: The PI3K-p110α kinase mutation bearing CRC cells were more motile as compared to the helical domain mutation bearing cells. This data supports our previous in vivo data that kinase domain mutations have a greater metastatic capability than helical domain bearing mutants. Cell motility was abrogated by both nonspecific and p110α directed PI3K inhibition suggesting that the activation of PI3K is necessary for CRC cell motility. This data supports the efforts to target the PI3K pathway in patients with advanced CRC.

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 470. doi:1538-7445.AM2012-470
Abstract 4708: Short-term romidepsin treatment combined with mitogen-activated protein kinase and phosphatidylinositol 3-kinase inhibition causes increased Bim expression and cell death in KRAS mutant cell lines
Authors: Bahr Julian C. , Robey Robert , Chakraborty Arup , Luchenko Victoria , Bates Susan E.
Published: August 2012

Romidepsin (FK228) is a histone deacetylase inhibitor (HDI) that modulates chromatin and activates apoptotic pathways in vitro. Clinical efficacy observed in peripheral and cutaneous T-cell lymphoma has not been replicated in solid tumors, as for other HDIs. We thus sought drug combinations that might improve the activity of romidepsin in solid tumors. Mutations in KRAS induce the mitogen activated protein kinase pathway (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathway. Laboratory data have suggested that activated MEK or AKT can confer resistance to HDIs. To determine if mitogen-activated protein kinase kinase (MEK) or AKT inhibitors could increase sensitivity to romidepsin, we measured cell death by annexin assay in a panel of 20 cell lines, nine KRAS mutants and eleven KRAS wild-type (WT). Cells were exposed to 25 ng/ml romidepsin for 6 h in the presence of 250 nM MEK inhibitor PD0325901 and/or 1 µM AKT inhibitor MK2206, after which romidepsin was removed and cells were incubated with the inhibitors alone for an additional 42 h. Among the 20 solid tumor cell lines examined, those harboring a KRAS mutation had a higher percentage of cell death than lines with WT KRAS when treated with the PD0325901/romidepsin combination (46.1±18% vs. 21.6±7.6% respectively, p-value .0057), the MK2206/romidepsin combination (43.8±15.8% vs. 28.6±13.6%, respectively, p-value .0470), or the PD0325901/MK2206/romidepsin combination (66.5±14.8% vs. 35.0±18.8%, respectively, p-value .0008). There was no statistically significant difference (p-value > .05) in cell death between KRAS and WT cell lines treated with any of the compounds alone. As expression of the pro-apoptotic protein Bim has been associated with romidepsin-mediated cell death, and as the MAPK pathway has been shown to phosphorylate and inactivate Bim, we examined Bim expression in cell lines sensitive to combined treatment with romidepsin and inhibitors. In the OVCAR5 cell line, which harbors a G12V KRAS mutation, we noted that romidepsin induced Bim expression and that combined treatment with PD0325901 led to higher expression by western blot. The high levels of Bim expression were associated with increased poly ADP ribose polymerase cleavage. These data demonstrate the benefit of combining an HDI with MEK and AKT inhibitors in KRAS mutant cell lines. Romidepsin and MAPK pathway inhibitor combinations should be further evaluated in patients with mutant KRAS cancers.

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 4708. doi:1538-7445.AM2012-4708
Abstract 4709: Combined mitogen-activated protein kinase pathway inhibition with short-term romidepsin treatment induces proapoptotic Bim and cell death in BRAF mutant cancers
Authors: Robey Robert W. , Luchenko Victoria L. , Bahr Julian , Chakraborty Arup R. , Zimmer Alexandra S. , Piekarz Richard L. , Bates Susan E.
Published: August 2012

Solid tumor trials with histone deacetylase inhibitors (HDIs) have been largely disappointing. We recently characterized a romidepsin-resistant T-cell lymphoma cell line that was found to have activation of the mitogen-activated protein kinase (MAPK) pathway, leading to subsequent phosphorylation and degradation of the proapoptotic protein Bim, suggesting that activation of this pathway may also confer resistance to romidepsin and other HDIs. The V600E BRAF mutation has been found in approximately 60% of melanomas and approximately 15% of colon cancers and leads to constitutive activation of the MAPK pathway. We thus hypothesized that combined treatment with romidepsin and BRAF or mitogen-activated protein kinase kinase (MEK) inhibitors may be effective in cancers that harbor the V600E BRAF mutation. To more closely simulate clinical administration of romidepsin, 11 V600E BRAF mutant cell lines (8 melanomas and 3 colorectal cancers) were exposed to 25 ng/ml romidepsin for 6 h after which romidepsin was removed, cells were incubated in fresh medium for an additional 42 h and subsequently apoptosis was measured by Annexin V and propidium iodide staining. Of the 11 cell lines, 10 exhibited significantly higher annexin staining after short-term romidepsin treatment (control 7.1% ± 3.8% vs. treated 34.7% ± 17.8%, p<0.001). Cells were subsequently treated with medium containing 25 ng/ml romidepsin with 250 nM of the MEK inhibitors AZD6244 (selumetinib) and PD0325901 or 1 µM of the mutant BRAF inhibitor PLX4032 (vemurafenib) for 6 h, after which the cells were incubated an additional 42 h in medium containing the inhibitors alone. Combined treatment with romidepsin and AZD6244 resulted in a significantly higher percent of annexin positive cells (p<0.05) in 9 of the 11 BRAF mutant cell lines examined. Immunoblot analysis demonstrated inhibition of the MAPK pathway at the concentrations used and that apoptosis was associated with increased expression of Bim, as well as increased poly (ADP-ribose) polymerase cleavage. Our results suggest that combined treatment with romidpesin and BRAF or MEK inhibitors may be useful in the treatment of cancers that express mutant BRAF.

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 4709. doi:1538-7445.AM2012-4709
Abstract 471: Acute activation of beta2-adrenergic receptor inhibits in vitro renal carcinoma cell migration through betaArrestin2-mediated regulation of p115RhoGEF
Authors: Ma Xiaojie , Zhao Yu , Daaka Yehia , Nie Zhongzhen
Published: August 2012

Metastatic renal cell carcinoma (RCC) is fatal with no current effective treatment. Molecular mechanisms underlying progression to metastatic RCC are not fully understood, and gene array studies have revealed increased expression of beta2-adrenergic receptors (beta2AR) and betaArrestin2 genes in RCC. We examined the regulation of RCC migration and invasion, two critical steps in the progression to the metastatic disease, following beta2AR activation using scratch assays and Boyden chamber assays in cultured RCC cell lines Caki-1, SN12C and RCC7. Acute treatment with the selective beta2AR agonist formoterol inhibited migration of the three cell lines by 40% to 70% with an IC50 of 2.5 nM or less. We further examined contribution of G protein and betaArrestin signaling. Using the betaAR-inverse agonist carvedilol that is biased for[[Unsupported Character - ]]betaArrestin signaling, combined with transient knockdown of Gs or betaArrestin2 by siRNA, or mouse embryonic fibroblasts from betaArrestin1 or betaArrestin2 knockout mice, we identified betaArrestin2 as a mediator of beta2AR-induced inhibition of cell migration. betaArrestin2 forms a complex with p115RhoGEF (guanine nucleotide exchange factor) that was previously shown to be regulated by Gq and G12/13. Activation of beta2AR promoted the translocation of p115RhoGEF to the plasma membrane, with consequent activation of RhoA which enhances formation of focal adhesions that restrain the cell migration. These results suggest that betaArrestin2 exerts cell compartment-specific effects on the cell migration. In the absence of acute beta2AR activation, betaArrestin2 sequesters p115RhoGEF in the cytosol, leading to relatively lower RhoA activity at the cell periphery and higher cell migration rate. Upon acute beta2AR activation, betaArrestin2 translocates to the plasma membrane with concomitant enrichment of p115RhoGEF at the plasma membrane, resulting in the activation of RhoA and decreased cell motility. Our results imply that betaArrestin2 and its effectors impact RCC metastasis and suggest its utility as a drug target to limit progression of the disease.

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 471. doi:1538-7445.AM2012-471
Abstract 4712: Target epigenetic regulation at a specific genomic region by using DNA binding molecules and HDACi
Authors: Nagase Hiroki , Watanabe Takayoshi , Koshikawa Nobuko
Published: August 2012

Pyrrole-Imidazole (PI) polyamide molecule was originally designed from structures of natural DNA binding molecule, such as Distamicine and Diocarbamicine and has been recognized as a synthetic molecule which binds the minor groove of Watson-Click base pair of double-stranded DNA in a sequence-dependent manner. We hypothesized conjugates between this synthetic DNA binding molecule and Histone Deacetylase inhibitor (HDACi) of Suberoylanilide hydroxamic acid (SAHA) should regulate histone acetylation in a specific region of the genome. We have taken two strategies to confirm this hypothesis. One is the candidate gene approach and another is chemical library approach. We synthesized a PI polyamide targeted the p16INK4a tumor suppressor gene and also synthesized a library of sequence-specific PI polyamides conjugated with the potent HDAC inhibitor, termed PIP-SAHA and screened their effect on the expression of tumor suppressor genes and Yamanaka factors, respectively. Chromatin immunoprecipitation analysis revealed SAHA-PIP induce acetylation of Histone H4 and H3 in the promoter region of p16, Oct-4 and Nanog. SAHA-PIP showed significant induction against c-Myc, Oct-3/4, Sox-2, and Klf-4, respectively in MEF but did not show significant induction of tumor suppressor p16 transcription. We also confirmed only SAHA did not show such histon acetylation and transcription induction, which implicated the role of PI polyamide conjugates in the induction of histone acetylation in specific genomic regions. Furthermore, a significant change in MEF morphology was demonstrated with our hit SAHA-PIPs. Those data suggests that a capability of PIP-SAHA regulate histone modification in a genomic sequence specific manner and promote agents for reprogramming efficiency in order to generate iPSCs, although the endogenous tumor suppressor gene expression was not observed, probably by means of “double lock” epigenetic silencing of tumor suppressor gene.

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 4712. doi:1538-7445.AM2012-4712
Abstract 4715: Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid affects apoptosis and tumor growth suppression of nasopharyngeal carcinoma
Authors: Chiang Alan KS , Hui Kwai Fung
Published: August 2012

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We examined the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, on EBV lytic cycle induction in NPC and investigated the cellular consequences. Micromolar concentrations of SAHA potently induced EBV lytic cycle with replication of EBV DNA, expression of immediate early (Zta and Rta), early (BMRF1) and late (gp350) lytic proteins and production of infectious viral particles in culture supernatants in three of four EBV-positive NPC cell lines. One NPC cell line had abortive EBV lytic cycle (no EBV DNA replication or late lytic protein expression) induction. Same concentrations of SAHA effected enhanced killing of EBV-positive NPC cells in comparison with EBV-negative counterparts, as measured by MTT assay. Increased percentages of annexin V-positive and TUNEL-positive cells and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC were also observed. Immunofluorescence staining and flow cytometry showed that the majority of NPC cells (>85%) expressing Zta, BMRF1 or gp350 lytic protein co-expressed cleaved caspase-3. Tracking of expression of EBV lytic protein and cleaved caspase-3 showed that proportion of NPC cells co-expressing EBV lytic protein and cleaved caspase-3 increased over time. Furthermore, knockdown of Zta expression by short-hairpin RNA significantly decreased proteolytic cleavage of caspase-3 in SAHA-treated EBV-positive NPC cells whilst overexpression of Zta induced apoptosis of EBV-positive NPC cells, confirming that lytic cycle induction programmed NPC cells to apoptosis. Interestingly, inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, suggesting that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were observed in NPC xenografts established in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA effects apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer. This project is funded by NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research) and Epstein-Barr virus research (# 20004525) grants of AKSC.

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 4715. doi:1538-7445.AM2012-4715
Abstract 4717: The anti-leukemic efficacy of the histone deacetylase inhibitor phenylbutyrate (PB): Reversal of epigenetic inactivation of WNT signalling pathway by PB
Authors: Miller Alexandra C. , Cohen Stuart , Rivas Rafael , Miller Karvelisse , Lison Paul
Published: August 2012

The histone deacetylase inhibitor (HDAC), phenylbutyrate (PB), is a novel antitumor agent. PB has shown anti-cancer efficacy in clinical trials and in in vitro and in vivo models. The anti-neoplastic effects have been linked to protein farnesylation inhibition and pro-apoptotic action in radiation-induced acute myeloid leukemia (AML) mice and malignant cells. Histone deacetylase (HDAC) is involved in epigenetic control of gene expression. The objective of the current study was to evaluate additional mechanisms i.e., epigenetic activity, of PB. A radiation late effects model was used to study PB epigenetic mechanisms. Mice were sub-lethally irradiated (3.5 Gy, 60Co) to induce leukemia. To test the anti-leukemia efficacy of PB and epigenetic mechanisms, mice were irradiated and then injected with PB (25 ug/kg) or saline daily 3 x weekly (2 weeks) for a total of 6 doses. Secreted Frizzled-related protein genes (SFRPs), which function in the Wnt signaling pathway, have been found to be down-regulated by promoter hypomethylation in human acute myeloid leukemia. Therefore, the methylation status of SFRP genes was analyzed in 20 samples obtained from radiation-induced AML mice. Aberrant CpG island methylation was observed in all four SFRP genes tested by methylation-specific PCR in AML mice. The frequencies of aberrant methylation among the irradiated mice samples were 55% for SFRP1, 30% for SFRP2, 15% for SFRP4, and 32% for SFRP5. In contrast, mice irradiated and treated with PB demonstrated aberration frequencies of 12% for SFRP1, 6% for SFRP2, 0% for SFRP4, and 4% for SFRP5 at 120 days post-radiation. At day 120 post-radiation, 50% of irradiated mice had developed leukemia while only 20% of mice treated with PB showed leukemia. The results demonstrate 1) that PB is an effective countermeasure against radiation-induced leukemia, 2) that epigenetic alterations are associated with radiation-induced leukemia and 3) that PB's efficacy may be associated with prevention of epigenetic disturbances involved in leukemogenesis. These studies do not reflect the opinion of the U.S. Government or the U.S. Department of Defense and are the scientific opinions of the author. This work was supported by DTRA Grant G1B2BJ, G2B2EM, AFRRI/USUHS RAB5AH, EU05-0089, and EU07-102.

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 4717. doi:1538-7445.AM2012-4717
Abstract 472: Nek3 kinase in breast cancer pathogenesis: The role of paxillin phosphorylation
Authors: Harrington Katherine , Clevenger Charles
Published: August 2012

Breast cancer progression to metastatic disease is responsible for the majority of breast cancer related deaths. The acquisition of a motile and invasive phenotype is a critical step in the metastatic process. The hormone prolactin (PRL) is increasingly recognized as contributing to the development and progression of human breast cancer. In particular, data from our laboratory has shown that PRL serves as a chemoattractant for breast cancer cells and can stimulate the formation of the focal-adhesion complex that is necessary for cell motility. PRL mediates its biological effects through binding to the PRL receptor (PRLr) and initiates several downstream signaling cascades, including activation of the serine/threonine kinase, Nek3. Nek3 regulates breast cancer cell survival, motility and invasion and has been shown to interact with the focal-adhesion adaptor protein, paxillin, in a PRL-dependent manner. Interestingly, we have determined that knockdown of Nek3 by siRNA significantly decreases the phosphorylation of paxillin, which suggests that paxillin may represent a novel substrate of Nek3. Given these findings, we sought to further elucidate the role of Nek3 in the regulation of paxillin phosphorylation and function in breast cancer. Co-immunoprecipitation analysis using mutant truncated proteins demonstrated that Nek3 interacts with the C-terminal region of paxillin, which contains 4 tandem LIM domains. Additionally, to determine the specific site(s) of phosphorylation an in vitro kinase assay was performed and it was determined that Nek3 directly phosphorylates paxillin at T401 located in the LIM2 domain. It is known that LIM2 and LIM3 comprise the focal adhesion targeting motif of paxillin and phosphorylation in this region plays an important role in modulating its activity. Ongoing studies will determine the biological effects of Nek3-mediated phosphorylation at paxillin T401 by examining the effect on breast cancer cell motility and invasion as well as the effect on paxillin localization and function in the focal adhesion complex. These studies will provide novel mechanistic insight into how Nek3-mediated phosphorylation of paxillin contributes to the pathogenesis of breast cancer. We propose that Nek3 is a potential novel therapeutic target for the treatment of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 472. doi:1538-7445.AM2012-472
Abstract 4723: HO-3867, a STAT3 inhibitor induces G2/M arrest and apoptosis in BRCA1-mutated ovarian cancer cells
Authors: Tierney Brent J. , McCann Georgia , Cohn David E. , Sudhakar Meryl , O'Malley David , Karuppaiyah Selvendiran
Published: August 2012

Background: BRCA1 plays an important role in DNA damage and repair, homologous recombination, cell-cycle regulation, and apoptosis. BRCA-mutated ovarian cancer often presents at an advanced stage, however, patients have shown improved responses with platinum-based chemotherapy versus sporadic cases. In spite of this, most patients will develop a recurrence and eventually succumb to the disease. Preclinical studies are currently investigating natural compounds and their analogs for tumor-directed targets in ovarian cancer cell models. The aim of this study is to investigate whether the STAT3 inhibitor HO-3867, a novel curcumin analog, has a therapeutic effect on BRCA1-mutated ovarian cancer. Methods: Expression of STAT family proteins, cell cycle, apoptotic and DNA repair proteins were evaluated via western blot on BRCA- mutated and non-mutated ovarian cancer cell lines. Cell viability and proliferation were analyzed with MTT and clonogenic assays. Cell cycle analysis and apoptosis assays were performed using flow cytometry and Annexin V staining. Reactive oxygen species (ROS) staining was performed using immunofluoresence. Finally, STAT3 overexpression or suppression experiments were performed using transfected wild-type STAT3 cDNA and STAT3 siRNA, respectively. Results: Our novel agent, HO-3867, and a commercial STAT3 inhibitor, STATIC, significantly inhibited BRCA-mutated cancer cells in vitro, in a dose-dependent manner. HO-3867 induced G2/M arrest with increased expression of p53 and p21 and with decreased cdk5 and cyclin D1 expression. BRCA-mutated cells treated with HO-3867 exhibited an increased induction of apoptosis with elevated levels of cleaved caspase-3, caspase-7, and PARP. Tyrosine-phosphorylated STAT3 (pTyr705) exhibited higher expression levels in BRCA-mutated cancer cells compared with other STAT proteins. Furthermore, treatment of these cells with HO-3867 resulted in decreased expression of pTyr705 and other DNA repair proteins. In addition, HO-3867 treatment induced more ROS in BRCA-mutated cells compared with wild-type cells, with no increased induction in benign ovarian surface epithelial cells. Furthermore, we confirmed that the over-expression of STAT3 cDNA provides resistance to HO-3867 induction of apoptosis. Conclusion: Our results show that HO-3867, a potential STAT3 inhibitor, may be effective in the treatment of BRCA-mutated ovarian cancer, although continued studies are needed to elucidate the exact mechanism of action.

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 4723. doi:1538-7445.AM2012-4723
Abstract 4725: Inhibition of the Hedgehog signaling pathway - a new target in treatment for children with neuroblastoma
Authors: Wickstrom Malin , Dyberg Cecilia , Shimokawa Takashi , Baryawno Ninib , Milosevic Jelena , Fuskevag Ole M. , Larsson Rolf , Zaphiropoulos Peter G. , Kogner Per , Johnsen John I.
Published: August 2012

Background: Neuroblastoma originates from neural crest cells and is one of the most common and deadly tumors of childhood. The majority of tumors develop therapy resistance with fatal clinical outcome despite intensive multimodal therapy. The Hedgehog (HH) signaling pathway is fundamental in embryonic development, controlling cell fate and proliferation. Activation of HH signaling is involved in several cancers, among them pediatric cancers. Studies have revealed that human neuroblastoma cells express high levels of proteins involved in HH signaling. GANT61 was recently identified as an inhibitor of GLI1 and GLI2, important transcription factors in the HH signaling pathway. The aim of this study was to investigate the impact of antagonists of HH signaling in neuroblastoma through characterization of the effects of small molecule inhibitors of the HH pathway, in vitro and in vivo. Methods: Cytotoxic activity of HH inhibitors was studied in cell viability and clonogenic assays. The molecular mechanisms were characterized using cell- and molecular biology techniques. Xenograft studies in mice were performed to validate the therapeutic effects and toxicity in vivo. Results: The cytotoxicity of different HH inhibitors (cyclopamine, SANT1 and GANT61) was evaluated in a panel of human neuroblastoma cell lines. GANT61 was the most potent drug tested with IC50 values in the range of 5.8 - 13 µM after 72h incubation. Expression of GLI1 significantly correlated to the log IC50 for GANT61 (R2=0.88), the higher the expression of GLI1 the lower the amount of GANT61 was required to inhibit cellular growth. We also saw an inverse correlation with MYCN (R2=−0.88). When measured with a GLI-dependent luciferase reporter plasmid, the GLI activity was decreased with 36% to 80% after GANT61 treatment (10 µM, 48h) depending on cell line. Investigation of cell cycle regulation and cell death demonstrated that GANT61 inhibited proliferation and induced apoptosis. siRNA experiments revealed that knockdown of GLI1 reduced proliferation more potently than GLI2, GLI3 and SMO inhibition. GANT61 significantly inhibited tumor growth in neuroblastoma xenografts in nude NMRI nu/nu mice without causing any sign of toxicity. Conclusions: These results suggest that inhibitors of the HH signaling pathway such as GANT61 may constitute a novel option for treatment to patient with high-risk neuroblastoma without MYCN-amplification.

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 4725. doi:1538-7445.AM2012-4725
Abstract 4731: Cytotoxic versus cytostatic effects in tumor models by spiro-oxindole-based modulators of the MDM2-p53 interaction
Authors: Meaux Isabelle , Barrière Cédric , McEachern Donna , Guerif Stéphane , Besret Laurent , Dos-Santos Odette , D'Agostino Stéphane , Sanchez Isabelle , Gorge-Bernat Dimitri , Pannier Pascal , Ratet Nathalie , Watters James , Miao Harry , Sager Jason , Lengauer Christoph , Garcia-Echeverria Carlos , Wang Shaomeng , Leopold Lance , Debussche Laurent
Published: August 2012

Two spiro-oxindole-based modulators -compounds A and B - of the MDM2-p53 protein-protein interaction display suitable pharmacological properties and are currently under advanced preclinical development (1). In vitro, these compounds induce massive cell death in a subset of relevant tumor models such as MDM2-amplified SJSA-1, while they can significantly block cell proliferation without detectable cytotoxic effect in other human cell lines (e.g. HCT116). To better characterize in vivo the different tumor cell sensitivity of these novel MDM2 antagonists, quantitative and longitudinal pharmacodynamic effects have been determined in several preclinical settings: i) anti-tumor efficacy, ii) activation of p53-dependent molecular biomarkers such as p53 itself, p21, MDM2, PUMA, Noxa, Bax and MIC-1 and iii) downstream biological effects monitored by immuno-assays as well as by in vivo fluorescence tomography and PET imaging. The results obtained from these studies have revealed striking differences in the biological read-outs associated with the observed cytotoxic versus cytostatic effects and provide guidance for the use of certain biomarkers in the clinical development of this new generation of anti-cancer agents. 1. Wang S. et al., AACR, Orlando FL, 2011, Abstract LB-204

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 4731. doi:1538-7445.AM2012-4731
Abstract 4736: N-myc downstream regulated gene 1: A novel mediator of hypoxia-driven chemoresistance in glioblastoma
Authors: Blaes Jonas , Weiler Markus , Sahm Felix , Pfenning Philipp-Niclas , Pusch Stefan , Jugold Manfred , Kempf Tore , Warnken Uwe , Weller Michael , Schnölzer Martina , Hartmann Christian , von Deimling Andreas , Wick Wolfgang
Published: August 2012

Like other solid tumors, glioblastomas (GB) feature vast hypoxic areas that are thought to promote tumor growth, angiogenesis and even drug resistance and hence facilitate an aggressive growth and recurrence after treatment. Elucidation of hypoxia-signalling and identification of the major regulators of hypoxia-dependent chemoprotection seems logical for the optimization of current treatment regimens. Using comparative 2D gel electrophoresis and mass spectrometry analysis, we here identify N-myc downstream regulated gene 1 (NDRG1) as upregulated by chronic sublethal hypoxia in GB cell lines. Immunohistochemical analysis of human astrocytoma specimens shows a marked increase in expression from WHO °II to °IV and a preferential perinecrotic localization of NDRG1 in GB. In vitro, besides the hypoxia-driven induction of NDRG1, which is mediated by Hif1α and Hif2α, NDRG1 is also induced by irradiation. NDRG1 limits glioma cell invasiveness in vitro and reduces proliferation in vitro and in vivo. Furthermore NDRG1 acts antiangiogenic with reduced endothelial cell migration and sprouting, and interferes with the expression of multiple angiogenesis-related signalling molecules. Most interestingly NDRG1 protects from temozolomide (TMZ)-induced G2/M-arrest and subsequent reduction of tumor cell proliferation. Analyzing pairs of GB tissue samples of relapsed glioblastoma patients reveals a dramatic increase of NDRG1-positive cells suggesting a treatment-related selection for NDRG1 expressing populations and a potential role for NDRG1 in the recurrence of GB. In line with these findings, quantifying the NDRG1 status in tissue specimens of the UKT05 GB trial, which looked at a dose-intensified TMZ therapy plus standard radiotherapy, reveals a negative correlation between NDRG1 status and overall survival. In the present work we identify hypoxia-driven NDRG1 as a modulator of tumor growth, invasion, angiogenesis and as a novel mediator of chemoresistance towards TMZ in GB. Our findings shed light on the role of hypoxia in adaption to chemotherapeutic treatment and introduce NDRG1 as a promising candidate for targeted therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4736. doi:1538-7445.AM2012-4736
Abstract 4738: The early response protein EGR-1 mediates vitamin E γ-tocotrienol-induced apoptosis in pancreatic cancer cells
Authors: Wang Chen , Pimiento Jose M. , Husain Kazim , Zhang Anying , Tong Zhongsheng , Malafa Mokenge P.
Published: August 2012

Background: Vitamin E tocotrienols are bioactive components of cereal foods such as oats, barley, rice bran, and palm, that have demonstrated anticancer and chemopreventive activities in various types of cancer. Recently we have shown that γ-tocotrienol is the most bioactive tocotrienol against pancreatic cancer both in vitro as well as in vivo. However, the molecular mechanism of action of tocotrienols in pancreatic cancer has not been fully elucidated. In this work, we investigated the early genes associated with Vitamin E γ-tocotrienol induced apoptosis, and therefore identify novel pathways related with its anticancer effects. Methods: Human pancreatic cancer MiaPaca-2 cells were treated with γ-tocotrienol (50 µM) and vehicle (ethanol) for 3 h and were subjected to microarray analysis using costume Affymetrix for the identification of early regulatory genes. Early growth response protein-1 (EGR-1) was one of those early response genes induced by γ-tocotrienol. Then we evaluated the protein expression of EGR-1 by real-time PCR and western-blot in pancreatic cancer cell lines (MiaPaca-2 and Panc-1) after treatment with γ-tocotrienol. Mechanistic studies were conducted trough siRNA knockdown of EGR-1 and the rescue of Vitamin of γ-tocotrienol-induced apoptosis, anti-proliferative effects and signaling pathways. Results: We identified 8 genes that were induced and 8 genes that were suppressed early after treatment with γ-tocotrienol in MiaPaca-2 cells. Then we selected and focused on the EGR-1 for signaling pathway analysis. Induction of protein expression was noticed in both MiaPac-2 cells and Panc-1 (6 fold and 10 fold respectively;p<0.05) after 24 h γ-tocotrienol treatment. γ-tocotrienol-induced apoptosis in MiaPac-2 cells and Panc-1 cells (PARP1 cleavage and Annexin V) was associated with induction of p-JNK, p-C-JUN, and EGR-1. Knockdown of EGR-1 in this two cell lines partially rescued γ-tocotrienol-induced apoptosis. Conclusion: Our data identifies EGR-1 as an early gene which is induced by Vitamin E γ-tocotrienol in pancreatic cancer cells. Activation of the EGR-1 pathway, may constitute a novel potential strategy for inhibiting pancreatic tumor growth.

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 4738. doi:1538-7445.AM2012-4738
Abstract 4739: miR-627 and histone demethylase JMJD1A as new therapeutic targets in colon cancer
Authors: Padi Sathish , Guo Bin
Published: August 2012

Vitamin D has excellent antitumor activities against various types of cancers, but it cannot be used for cancer therapy due to its side effect of causing hypercalcemia. To understand its mechanism of action in tumor suppression, we investigated the effects of calcitriol (1α,25-dihydroxyvitamin D3, the active form of vitamin D) on microRNA expression in colon cancer cells. We identified miR-627 as the sole microRNA whose expression was stimulated by calcitriol. Furthermore, JMJD1A (jumonji domain containing 1A), the gene encoding a histone demethylase, was identified as the target of miR-627. By downregulating JMJD1A, miR-627 mediated the effects of calcitriol to increase histone H3K9 methylation and to suppress the expression of growth-promoting genes, such as GDF15. Calcitriol also induced miR-627 and downregulated JMJD1A in colon cancer xenografts in nude mice. Overexpression of miR-627 decreased JMJD1A and suppressed colon cancer growth both in vitro and in vivo in the nude mice. In addition, we found that miR-627 expression was decreased in human colon cancer specimens when compared to its levels in the normal colon tissues. Thus, miR-627 plays an important role in the epigenetic action of vitamin D in tumor suppression. miR-627 and JMJD1A may serve as potential targets to exploit the antitumor activity of vitamin D without eliciting its hypercalcemic side effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4739. doi:1538-7445.AM2012-4739
Abstract 474: The essential role of Giα2 in prostate cancer progression
Authors: Zhong Miao , Clarke Shineka , Khan Shafiq A.
Published: August 2012

Basic and clinic research data have suggested that signaling of G-protein-coupled receptors (GPCRs) is amplified in malignant prostate tumors and facilitates the transition of primary cancer cells to the androgen independent stage. Oxytocin (OXT) is a neurohypophyseal peptide hormone that regulates normal prostate functions. However, the information is very limited about the effect of OXT on prostate cancer progression. The effect of OXT is mediated by a GPCR that promiscuously activates multiple G-proteins including Gqα and Giα. Recently, we have shown that OXT receptor (OXTR) is the primary target of OXT in androgen independent prostate cancer cell lines (DU145 and PC3). Moreover, activated OXTR has been shown to promote migration of PC3 cells via a pertussis toxin (PTx) sensitive Gi/o-protein dependent mechanism. In the present study, OXTR was shown to be increased in advanced prostate cancer tissues when compared with normal prostate tissues. Expression of OXTR protein is detected in DU145 and PC3 prostate cancer cell lines but not in the immortalized prostate epithelial cells (RWPE1). OXT treatment (100 nM) inhibited the foskolin (5 µM) induced cAMP accumulation in PC3 cells, as indicated by the cAMP luciferase reporter assay. This data has provided direct evidence that supports the involvement of Giα in the OXTR signaling in PC3 cells. There was no difference in expression of Giα isoforms (Giα1-3) at the mRNA level between normal (PrEC, RWPE1 and RWPE2) and prostate cancer cell lines (LNCaP, DU145, PC3, and PC3M). However, western blot analysis showed that Giα2 is the only Giα family member detectable in PC3 cells. PTx (100 ng/ml) has been shown to abolish the OXT-induced migration of PC3 cells. Overexpression of PTx resistant Giα2, but not wild type Giα2, restored the effect of OXT in PTx pretreated PC3 cells. Accordingly, genetic silencing of Giα2 expression by siRNA eliminated the OXT-induced migration of PC3 cells. Unexpectedly, knock down Giα2 also inhibited the EGF (3 ng/ml) induce migration of PC3 and DU145 cells. In conclusion, Giα2 may play an essential role in prostate cancer metastasis.

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 474. doi:1538-7445.AM2012-474
Abstract 4741: Development of a novel therapeutic agent for breast cancer based on a new pathway analysis of triple-negative breast cancer
Authors: Fujimori Minoru , Fujita Tomoyuki , Mizukami Tamio , Okawara Tadashi , Inoue Kengo
Published: August 2012

We developed a novel method for drug discovery using active compounds as pharmacodynamic biomarkers in triple-negative breast cancer cell lines. [Materials and Methods] Compounds that selectively inhibit proliferation of triple-negative breast cancer were screened in triple-negative breast cancer cell lines (MDA-MB231, MDA-MB468 and HCC-38) and control MCF7 and HeLa cell lines. A small molecule library was designed to seek structural diversity by chemical informatics and a total of 30,000 synthetic compounds were assessed considering drug similarities. The differences in gene expression profiles in the cell lines between before and after the addition of the selected compounds were analyzed and compared with the control cells. [Results] Seven compounds that exhibited inhibitory effects on the triple-negative breast cancer cell lines were found in the compound library. These compounds exhibited inhibitory activity with IC50-values ranging from 1.2 to 10.8 μM. The results of the microarray analysis indicated several pathways including the irinotecan pathway that changed specifically in the triple-negative breast cancer cell lines by the addition of the compounds.[Conclusion] A research strategy to screen a compound library with structural diversity and develop a drug discovery system using active compounds as pharmacodynamic biomarkers can be widely applied as a new approach for developing innovative personalized medicine.

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 4741. doi:1538-7445.AM2012-4741
Abstract 4744: Development of an immunoproteasome fluorescent substrate: A functional proteomics tool
Authors: Lee Na-Ra , Carmony Kimberly C. , Wu Ying , Miller Zachary , Kim Kyung-Bo
Published: August 2012

Introduction: This research was conducted to develop the first immunoproteasome fluorescent substrate. The immunoproteasome is an alternate form of the constitutive proteasome with distinct catalytic subunits. While immunoproteasomes appear to generate peptides for MHC class I antigen presentation, their functions are still not completely understood. Antibody-based studies have shown that immunoproteasome catalytic subunits are expressed in a variety of cancers, suggesting they may play important roles in this disease. However, antibodies bind both active and inactive subunits, rendering these studies incapable of correlating immunoproteasome activity with disease progression. Although fluorescent peptide substrates are widely used to monitor proteasome activity, those currently available are cleaved by both forms of the proteasome. Therefore, fluorescent substrates selectively cleaved by the immunoproteasome are needed to correlate their activity with cancer progression. Experimental Procedures: The fluorescent substrate was synthesized by replacing the epoxyketone pharmacophore of the immunoproteasome-selective inhibitor YU-102 with a 7-amino-4-methylcoumarin (AMC) fluorogenic moiety. The rates of hydrolysis of the resulting substrate, YU-102-AMC, by both purified constitutive proteasomes and immunoproteasomes were evaluated by monitoring the release of AMC over time. Hydrolysis of this substrate was also investigated in T1 and T2 cell lysates. Data Summary: The structure of the fluorescent substrate YU-102-AMC was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. YU-102-AMC was preferentially cleaved by purified immunoproteasomes in comparison with purified constitutive proteasomes. This preferential cleavage by immunoproteasomes was confirmed in lysates of cancer cell lines T1 and T2. Conclusions: We have successfully developed YU-102-AMC, an immunoproteasome fluorescent substrate which facilitates quantification of immunoproteasome activity in cancer cell lysates. This substrate will be utilized to examine a correlation between immunoproteasome activity and cancer progression.

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 4744. doi:1538-7445.AM2012-4744