bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 648: Combined PI3K and MEK inhibition in patient-derived xenografts generated from recurrent triple negative breast cancer
Authors: Ibrahim Yasir H. , Serra Violeta , Scaltriti Maurizio , Prudkin Ludmila , Anton Pilar , Perez José , Balmaña Judit , Baselga José
Published: June 2012

Both the PI3K and ERK pathways have become critical therapeutic targets due to their importance in breast cancer. Some triple negative breast cancers (TNBC) are sensitive to MEK inhibition due to the presence of RAS activation signatures. However, hyperactivation of PI3K signaling through PTEN loss impairs efficacy of MEK inhibitors in TNBC. On the other hand, it is likely that inhibition of PI3K in TNBC results in compensatory feedback activation of MEK, as previously observed in HER2 positive cell-line models. Therefore, combined inhibition of both PI3K and MEK would translate to optimal therapeutic efficacy in the clinic. We generated patient-derived xenografts (PDXs) of TNBC from patients with recurrent disease to assess the efficacy of this combined therapy in a model that more closely resembles the clinical scenario.

Upon completion of informed patient consent, biopsies from patients with cutaneous, axillary or subclavicular metastases that had progressed from standard therapies were implanted in female nude mice. Five TBNC PDXs were expanded and confirmed to have equivalent pathological and morphological features as the original patient samples; they maintained similar ER, PR, HER2, PTEN and Ki67 status. Mutation status of PI3K, PTEN and RAS pathway were also evaluated. Additionally, the PTEN-loss transcriptional program and RAS-like transcriptional program were evaluated to further classify the tumors. PDXs were treated with a pan-class I PI3K inhibitor (NVP-BKM120) and a MEK inhibitor (AZD6244) as single agent or in combination. Preliminary tumor growth studies in a PDX that lacked PTEN expression demonstrated NVP-BKM120 sensitivity, but no response to AZD6244. The combination of NVP-BKM120 plus AZD6244 augmented the PI3K tumor inhibitory effect. Ongoing studies will more precisely delineate the relationship between pathway activation status and pharmacodynamic response to inhibitors targeting PI3K or MEK. These data highlight the great potential for evaluating new strategies in models that truly represent breast cancer of patients enrolling in clinical trials.

In these studies we attained data from models that faithfully resemble the disease observed in the clinic supporting the advancement of clinical trials targeting both PI3K and Ras/Raf pathways in TNBC. Since these models strongly resemble the clinical disease, PDXs can also offer new opportunities to test experimental therapies that are normally hindered by the homogeneity and culture selection of standard cell-lines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2011-648
Abstract 650: Ex vivo analysis of the mTOR/PI3K & MEK/ERK inhibitors, BEZ235 & AZD6244, alone and in combination in human tumor primary culture micro-spheroids: Exploration of horizontal pathway targeting
Authors: Nagourney Robert Alan , Bernard Paula J. , Francisco Federico R. , Wexler Ryan , Evans Steven S.
Published: June 2012

Therapeutic targeting of signal transduction pathways offers fertile ground for the discovery of small molecule kinase inhibitors and novel combinations. The success of Imatinib in CML, the first clinically approved oral TKI, reflects in part, the linear association between the malignant phenotype and c-Abl activation. However, most solid tumors reveal complex interactions between signal pathways that cross talk at points of commonality, the subject of prior report (Nagourney, RA Proc. AACR, 2010). To examine the clinical potential of BEZ235 and AZD6244, inhibitors of the PI3K & MEK/ERK pathways, we applied Ex Vivo Analysis of Programmed Cell Death (EVA/PCD™) (Nagourney RA, Curr. Treat Options in Oncology, 2006) to tumor micro-spheroids isolated from 24 patients. Drugs were tested alone and in combination. Five point dose response curves were interpolated to provide LC50 values. Synergy was assessed by median effect. Activity comparisons were conducted by modified Z-Score. LC50 values by Z-score for AZD revealed Melanoma > Breast > NSCLC > Myeloma >Ovarian > Uterine >Sarcoma > Renal, while BEZ revealed Breast > Renal > NSCLC > Ovarian > Myeloma > Sarcoma > Uterine. By combining BEZ with AZD, we then explored the simultaneous inhibition of the MEK/ERK and PI3K/mTOR pathways. Results in 5 of the first 6 analyses revealed activity & synergy for the dual pathway inhibitor combination. These results support the clinical relevance of targeting the MEK/ERK and PI3K/mTOR pathways and more importantly suggest dual pathway inhibition (horizontal) to be a productive strategy for further clinical developmental. Disease specific profiles and sequence dependence are being explored and will be reported. Supported in part by the Vanguard Cancer Foundation and The Nagourney Institute.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 650. doi:10.1158/1538-7445.AM2011-650
Abstract 672: PBI-1402, a first-in-class erythropoiesis regulating agent, inhibits tumor growth and metastasis of murine mastocytoma and does not overshoot hemoglobin: Comparison with erythropoietin
Authors: Gagnon Lyne , Sarra-Bournet François , Geerts Lilianne , Hince Kathy , Lagraoui Mouna , Laurin Pierre , Penney Christopher , Grouix Brigitte
Published: June 2012

BACKGROUND: PBI-1402 reduces the need for transfusion and increases hemoglobin (Hb) level and red blood cell count (RBC) in chemotherapy-induced anemia (CIA) by a mechanism of action distinct from erythropoietin (EPO).

AIM: The objective was to compare the effect of PBI-1402 and EPO on modulation of tumor growth.

METHODS: The effect of PBI-1402 (200 mg/kg, oral, once a day) or EPO (200 or 2000 U/kg, s.c. injection, twice a week) was studied in a subcutaneous P815 mastocytoma model. P815 cells express both PBI-1402 and EPO receptors. Tumor growth, metastasis, serum nitric oxide (NO) and hematocrit (Ht) were assessed.

RESULTS: P815 tumor growth is rapid and metastasizes to the liver. Inflammation is also associated with P815 tumor growth. PBI-1402 significantly reduced tumor growth (T/C=37%) and invasion as demonstrated by a 33% reduction of mice with liver metastasis. In comparison, EPO had no effect (200 U/kg, equivalent dose used in CKD treatment). However, high dose EPO (2000 U/kg, equivalent dose used in human cancer treatment) exacerbated tumor growth (T/C: 135%). Both doses of EPO induced a significant increase in the percentage of mice with liver metastasis (2X, 200 U/kg, and 3X, 2000 U/kg) compared to control. PBI-1402 treatment had no effect on NO and did not overshoot normal hematocrit. However, treatment with high dose EPO significantly increased NO and Ht levels (Table 1).

CONCLUSION: These results suggest that oral treatment with PBI-1402 may inhibit growth and metastasis of cancer cells. As an important safety feature, PBI-1402 does not induce overshoot of hemoglobin. In contrast to EPO, PBI-1402 may offer the advantage of reducing tumor growth and metastasis while preventing anemia induced by chemotherapy. In conclusion, PBI-1402 has the potential to be a safe erythropoiesis-regulating agent useful in treating anemia in cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 672. doi:10.1158/1538-7445.AM2011-672
Abstract 680: Antitumor activity of CDDP and nuclear receptor PXR
Authors: Kishimoto Shuichi , Fukushima Shoji
Published: June 2012

The goal of this study was to evaluate the effect of the possibility of compound that regulate the function of nuclear receptor as agent enhancing the activity of CDDP. Cell lines used in this study were HepG2 and some CDDP-resistant HepG2 cells that were established in our laboratory. These CDDP-resistant HepG2 cells show IC50 values several times higher than that of HegG2 cells. Changes in gene expression were assessed in RT-PCR assays, and the ability of apoptosis induction was assessed as caspase-3 activity. Gene expressions of nuclear receptors in HepG2 cells after exposure to CDDP were examined over time. Interestingly, IC90 dose of CDDP induced significant reduction in PXR mRNA expression with remarkable apoptosis, but IC50 dose of CDDP did no apoptosis and no significant change in PXR mRNA expression. Also, six established CDDP-resistant HepG2 cells show increased PXR mRNA expression relative to parent HepG2 cells. Therfore, PXR agonist and antagonist were used to elucidate if PXR could modulate the sensitivity of cells to CDDP. HepG2 cells exposured to PXR agonist rifampicin for 24 hr showed increased PXR mRNA expression. In HepG2 cells, pre-exposure to rifampicin followed by exposure to CDDP and coexposure to rifampicin and CDDP reduced induction of apoptosis compared to exposure to CDDP alone. Moreover, almost total suppression of apoptosis induced by CDDP was achieved by pre-exposure for 48 hr followed by coexposure to rifampicin for 24 hr. PXR antagonist leflunomide caused a slight enhancement in apoptosis induced by CDDP in HepG2 cells and a significant enhancement in apoptosis induced by CDDP in CDDP-resistant HepG2 cells. We concluded that nuclear receptor PXR is an important factor which regulates the expression of genes that affect on the antitumor activity of CDDP and that PXR antagonist can be a candidate as an agent augmenting the antitumor activity of CDDP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2011-680
Abstract 683: Biological significance of conjugated linoleic acid in ovarian carcinoma
Authors: Shahzad Mian M. , Al-Niaimi Ahmed , Felder Mildred A. , Kapur Arvinder , Claussen Nick , Jallow Fatou , Van Galder Hannah R. , Connor Joseph P. , Patankar Manish S.
Published: June 2012

Background: Conjugated linoleic acid (CLA) has long been recognized to exert protective effects on carcinogenesis in breast, prostate and colon malignancies. Recent data suggests that CLA plays a key role in inhibiting several important survival pathways in breast cancer cell. However, data on effects of CLA in epithelial ovarian carcinoma are lacking and was the focus of this study.

Methods: A2780 and SKOV3 ovarian cancer cell were treated with increasing concentrations of two different isomers of CLA (9:11, 10:12) and effect of CLA on cell viability was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points with and without oxaliplatin. Additionally, effect of CLA isomer 10:12 were evaluated on A2780 ovarian cancer cell apoptosis by flow (Annexin V/PE).

Results: CLA isomer-10:12 significantly affected the ovarian cancer cell viability in a time and dose dependent fashion. Seventy-two to 96h of CLA treatment (isomer 10:12, 7µμ) resulted in ∼50% reduction in cell viability compared to control (p<0.01). A 20 µM dose of the CLA isomer 10:12 resulted in >80% reduction in live cell compared to controls. However, CLA isomer 9:11 had no effect in all doses and time points tested (0.001 µM- 50mM). Additionally, at IC50 dose of oxaliplatin treatment, the addition of CLA, resulted in a substantial reduction (by ∼50%, p<0.05) in percent live cell compared to oxaliplatin monotherapy.

Conclusion: Our data indicate that CLA can significantly hamper the survival of ovarian cancer cell. These findings may have therapeutic implications for ovarian cancer management and needs further exploration.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2011-683
Abstract 692: Drug treatment causes evolution
Authors: Flach Edward , Fedorenko Inna , Paraiso Kim , Smalley Keiran SM , Anderson Alexander RA
Published: June 2012

A targeted drug treatment, PLX40720, eliminates the bulk of melanoma tumours, but there is always regression. We assert that the tumour population is heterogenous. Then the drug treatment is a selection process, targeting specific subpopulations. When the treatment ends, phenotypic drift causes reversion towards the original wild-type population.

Our model is a discrete population of cells. The cells each have a distinct phenotype. This phenotype determines their fitness. The fitness changes under drug conditions: we define a fitness landscape for both drug and drug-free conditions. We validate the model using population experiments on four melanoma cell lines (1205Lu, WM164, WM793, WM983A). We find initial growth curves, death rates and regrowth curves under drug treatment.

Matching the clinical approach, we treat the cell lines chronically with drug for six months. We then stop treatment and examine the reversion of the resistant cell lines to wild-type over a period of several weeks. There is only partial reversion to wild-type. We return to the model to incorporate this experimental data. We extend the complexity of the fitness landscape to multiple fitness “wells". Reversion after drug treatment only populates one of these wells. This more complex landscape reproduces the experimental observations.

The model concept extends naturally to considering combination therapies. We also discuss the effect of modifying the local environment of the tumour: the drug treatment may become more effective. This approach gives a quantitative assessment of treatment strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 692. doi:10.1158/1538-7445.AM2011-692
Abstract 7: PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphotidylinositol 3-kinase pathway
Authors: Desai Shraddha , Acevedo-Duncan Mildred
Published: June 2012

Robust expression of atypical protein kinase C-iota (PKC-ι) is a hallmark of human glioma, benign and malignant meningiomas [R. Patel, et. al. (2008) Cell Prolif. 41, 122-135]. The phosphoinositide (PI) 3-kinase pathway is frequently found to be over stimulated due to activating mutations in growth factor receptors or loss of function of the tumor suppressor PTEN. Protein kinase C-ι is often activated by the PI 3-kinase pathway and is an essential downstream targeted mediator. We investigated the role of PKC-ι in two human glial tumor derived cell lines T98G and U87MG. PKC-ι directly phosphorylated and inactivated Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family at three phospho specific residues, Ser-112, Ser-136 and Ser-155. PKC-ι was also observed to co-localize and directly associate with Bad in the cytoplasm of the cells (as shown by Immunofluorescence, Immunoprecipitation, and Western blotting). Furthermore, purified, active PKC-ι induced direct phosphorylation of Bad at all three serine residues subsequently disrupting Bad/Bcl-XL dimerization. Reduction of PKC-ι upon RNA interference exhibited a corresponding reduction in Bad phosphorylation as did Wortmannin and LY294002, known PI3-Kinase inhibitors. Depletion of PKC-ι by siRNA treatment also induced apoptosis with release of cytochrome C, caspase 3 activation and PARP cleavage. These data provide evidence for novel PI3-kinase/PKC-ι/BAD cell survival pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2011-7
Abstract 723: Acquired resistance to PLX4720 involves both ERK re-activation and suppression of BH3-only proteins in human melanoma cells
Authors: Shao Yongping , Aplin Andrew E.
Published: June 2012

B-RAF is mutated to a constitutively active form in 8% of human cancers. Tumors harboring mutant B-RAF are often dependent on B-RAF-MEK-ERK1/2 signaling for the maintenance of malignant properties. Evidence for oncogene addiction has been underscored in early phase clinical trials in which the RAF inhibitor, PLX4032/RG7204, caused partial or complete responses in 81% of mutant B-RAF harboring melanoma patients. However, the average duration of response was 7-8 months with the majority of patients relapsing, indicating the acquisition of resistance to PLX4032. Since understanding the mechanisms of resistance will drive the design of future combinatorial trials, we undertook an in vitro analysis of melanoma cell acquired resistance to PLX4720, the tool compound for PLX4032. Through continuous culture in the presence of PLX4720, we developed mutant B-RAF melanoma cell populations that display resistance to B-RAF inhibitors. Resistance was associated with a partial re-activation of ERK1/2 signaling, recovery of G1/S cell cycle events, and suppression of BH3-only proteins, Bim-EL and Bmf, in the presence of PLX4720. Preventing the ERK1/2 re-activation with MEK inhibitors blocked G1-S cell cycle events but failed to induce apoptosis or up-regulate Bim-EL and Bmf levels. These data indicate that acquired resistance to PLX4032/4720 likely involves ERK1/2 pathway re-activation and ERK1/2-independent silencing of BH3-only protein up-regulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 723. doi:10.1158/1538-7445.AM2011-723
Abstract 76: Epigenetic silencing of argininosuccinate synthetase renders human bladder cancer cells sensitive to pegylated arginine deiminase
Authors: Allen Michael , Syed Nelofer , Luong Fiona , Howarth Kimberley , Gorman Patricia , Tomlinson Ian , Roylance Rebecca , Lu Yong-Jie , Nicholas Lemoine , Dan Berney , Crook Tim , Szlosarek Peter W.
Published: June 2012

Tumors deficient in argininosuccinate synthetase (ASS1), a key enzyme involved in arginine synthesis, are auxtrophic for arginine and sensitive to amino acid deprivation. Based on reports of instability at 9q34 in bladder cancer, the locus of ASS1, we investigated ASS1 status using a variety of molecular and genetic techniques and assessed the impact of arginine deprivation on the viability of bladder cancer cell lines in vitro and in vivo. Initially, we revealed very low mRNA levels and no ASS1 protein in 3 of 7 bladder cancer cell lines (253J, EJ28 and UMUC-3). The ASS1-negative cell lines were all hypermethylated at the ASS1 promoter, compared with the ASS1-positive cell lines, which were hypomethylated. There was no evidence for ASS1 mutations by gene sequencing or loss of heterozygosity by fluorescence in situ hybridization and single nucleotide polymorphism arrays. An analysis of 48 patients with bladder cancer revealed 65% were ASS1-negative by immunohistochemistry, with evidence for ASS1 methylation as the mechanism for ASS1 inactivation. The arginine depleting agent, pegylated arginine deiminase (ADI-PEG20), triggered caspase-dependent and independent apoptosis of 253J and EJ28 cell lines, respectively, whereas ASS1-positive control bladder cell lines were resistant to ADI-PEG20. We have identified downregulation of ribonucleotide reductase M2 (RRM2) as a biomarker of ADI-PEG20 activity and ongoing work is focused on combining ADI-PEG20 with cisplatin-doublet chemotherapy in vivo. In summary, we propose that epigenetic silencing of ASS1 renders bladder cancer sensitive to ADI-PEG20, providing a novel approach for the treatment of a common and therapeutically challenging cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 76. doi:10.1158/1538-7445.AM2011-76
Abstract 762: ONT-10, a liposomal vaccine targeting hypoglycosylated MUC1, induces a potent cellular and humoral response and suppresses the growth of MUC1 expressing tumors
Authors: Pestano Linda A. , Christian Brenda , Koppenol Sandy , Millard Jeff , Christianson Gary , Klucher Kevin , Rosler Rob , Peterson Scott R.
Published: June 2012

MUC1 is highly expressed in multiple tumor histologies and is hypoglycosylated relative to normal tissue, with prematurely terminated carbohydrate modifications including αN-acetylgalactosamine (Tn). The aberrant glycosylation state of MUC1 makes this antigen immunologically distinct in tumor relative to normal tissues and an attractive vaccine target.

ONT-10 is a liposomal formulated vaccine that incorporates a synthetic glycopeptide antigen that includes two complete 20 amino acid tandem repeats from human MUC1, including six glycosylated sites modified by Ser- or Thr-O- Tn, and is incorporated into the liposomal drug product via two C-terminal lipid serines. The ONT-10 drug product is formulated with the novel, fully synthetic lipid A adjuvant, penta erythritol lipid A (PET Lipid A). PET Lipid A displays a similar spatial distribution of lipid chains as natural Lipid A, but is composed of pure 6-acyl moieties, resulting in enhanced potency as an activator of Toll-like Receptor 2 and 4 (TLR2, TLR4).

In preclinical studies, immunization of mice with ONT-10 induces a prominent T cell and antibody response to MUC1 and results in a substantial growth inhibition of MUC1 expressing tumors. T cell recall assays show a Th1 cytokine pattern with strong IFNγ production in spleen derived CD4 and CD8 cells in response to Tn-MUC1. At doses as low as 5μg/mouse ONT-10 results in a high titer anti-MUC1 antibody response composed principally of IgG2b and IgG3 that show preferential binding to Tn-MUC1 and selectively target tumor cells that express human MUC1. In a preventative B16-MUC1 tumor model, vaccination with 5μg ONT-10 potently suppresses tumor growth, resulting in 99% mean tumor growth inhibition relative to controls with 9/12 animals tumor free four weeks after tumor cell implantation. Similarly, in a MC38-MUC1 tumor model 5μg ONT-10 results in an 90% reduction in tumor growth relative to controls. Based on these preclinical data, we propose that the combination of the glycopeptide antigen structure and improved Lipid A adjuvant in ONT-10 offer advantages relative to previous MUC1 targeted vaccines by potently activating both humoral and cellular immunity against hypoglycosylated MUC1 and that these features may provide additional therapeutic benefit to patients with MUC1 expressing cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 762. doi:10.1158/1538-7445.AM2011-762
Abstract 779: Role of CD44 in LAK cell-mediated killing of melanoma
Authors: Mckallip Robert J. , Law Gabriela , Sun Jingping
Published: June 2012

Lymphokine-activated killer cells play an important role in various immunologically-based treatments of cancer, including in the use of IL-2 therapy for the treatment of malignant melanoma. In previous studies, we demonstrated that the expression of CD44 on splenocytes is significantly elevated following exposure to IL-2. Therefore, in the current study we examined the potential significance of CD44 expression on lymphokine activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells and NKT cells. Treatment of melanoma bearing CD44WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma bearing CD44KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2 treated CD44KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2 treated CD44WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred IL-2-activated splenocytes from CD44KO mice were significantly less effective at controlling tumor growth in vivo. Next the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44lo) or elevated (CD44hi) expression of CD44 are generated and that the CD44hi cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 779. doi:10.1158/1538-7445.AM2011-779
Abstract 804: Class II transactivator (CIITA) transcription is reduced by the ER-E2 signaling pathway in breast cancer cells
Authors: Mostafa Ahmed , Codner Dianne , Lewis Christa , Hirasawa Kensuke , Christian Sherri , Steimle Viktor , Drover Sheila
Published: June 2012

The immune response is regulated by the interaction of CD4+T-cells with human leukocyte antigen class II (HLA-II). HLA-II genes code for HLA-DR that display peptides from antigens, which have undergone proteolysis in the endocytic pathway. Peptide loading of HLA-DR molecules also requires the co-chaperones; Invariant chain (Ii) and HLA-DM. Regulation of HLA-II occurs mainly at the transcription level, through interaction of transcription factors with the class II transactivator (CIITA). We previously reported that discordant expression of HLA-II in breast cancer associated with decreased estrogen receptor (ER) levels and younger age at diagnosis, suggesting that the estrogen-ER signaling pathway is implicated in regulating HLA-II in breast carcinoma cells. Moreover, using an experimental model of ER+ and ER− breast cancer cell lines, we found that estradiol (E2) augmented expression in the ER− cell line but decreased expression in the ER+ transfectant. To address whether ER and E2 interfere with CIITA expression, we performed Western blotting and quantitative real-time PCR in the same experimental cell system. CIITA expression was markedly reduced in ER+, but not in ER− cells; furthermore, CIITA inversely correlated with ER levels. CIITA promoter activity was analysed using a luciferase reporter gene construct in cell lines, treated or not with E2 and/or ICI 182,780 (ER antagonist). CIITA luciferase activity was reduced in ER+ cells and was further inhibited by E2 and was restored by ICI. This suggests that ER may be recruited to the CIITA promoter and indirectly interfere with transcription of HLA-II genes. We further analyzed HLA-II in a wild type ER+ line, MCF-7, which was stimulated with IFN-γ or stably transfected with CIITA. HLA-DR surface expression, ascertained by flow cytometry, was upregulated in cells grown in E2-depleted medium. Western blotting confirmed that ER degradation by ICI in MCF-7 correlated with up regulation of CIITA and HLA-II. In aggregate, these results indicate that the E2-ER pathway interferes with CIITA and HLA-II expression. Investigations such as this should improve our understanding of how ER and estrogen modulate the antigen presentation capabilities and immune recognition of breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2011-804
Abstract 806: Anti-tumor effects of low temperature whole body hyperthermia on human bladder cancer cells in vivo
Authors: Yamamoto Yutaka , De Velasco Marco A. , Wang Yi , Izumi Ayaka , Okazaki Erina , Doi Makiko , Hatanaka Yuji , Shimizu Nobutaka , Yoshimura Kazuhiro , Nozawa Masahiro , Yoshikawa Kazuhiro , Uemura Hirotugu
Published: June 2012

As a whole, the concept of thermotherapy is still under study. However, there is an abundant amount basic and pre-clinical published reports that have shown the antitumor effects of hyperthermia in various cancer models. Local, regional and whole body hyperthermia (WBH) has been studied and the effectiveness of the treatment depends largely on the temperature achieved and the duration of treatment. High temperatures, low duration (heat shock) will have a direct killing effect on exposed cells. The limitation to this approach is the restriction of tumor location. Low temperature, high duration WBH is generally considered to be the least toxic but requires long exposures that are generally uncomfortable or inconvenient to the patients. In this study we investigate the therapeutic efficacy of a synthetic far-infrared emitting biomaterial (BR), capable of inducing mild whole body hyperthermia, alone or in combination with chemotherapy against bladder cancer cell lines in vivo.

Immunocompetent and immunodeficient mice were inoculated with a human superficial bladder cancer cell (KU7/GFP) or mouse bladder cancer cell (NBT2), respectively, and treated with BR alone or in combination with doxorubicin (Dox). Mice exposed to BR increased their core body temperature by 1oC (P<0.05) after 2 weeks of exposure. Both immunocompetent and immunodeficient bearing subcutaneous tumor xenografts treated to WBH demonstrated a clear tendency in the reduction of tumor size. Histological analysis showed that tumors exposed to WBH had remarkable granulocyte infiltration accompanied by increased necrosis. Elevated levels of HSP27, HSP70 and HSP90 were detected in tumors of mice exposed to WBH. Increased levels of C-GSF, GM-CSF, TNFα and IL-12p70 corresponded to exposure to WBH. To determine the synergistic effects of WBH and chemotherapy, a survival study was carried out using immunodeficient mice orthotopically inoculated with KU7/GFP cells. Mice treated with Dox or WBH/Dox demonstrated a reduction in tumor size compared to mice treated with WBH and untreated controls. More interestingly, survival rates for control, WBH, Dox and WBH/Dox groups were 50% (5/10), 70% (7/10), 10% (1/10) and 50% (5/10), respectively. No clear differences were noted in HSP expression or granulocyte infiltration in these mice.

Our data suggests that WBH may induce mild antitumor effects; although, the precise mechanism is still unclear, it appears that it may be due to an immuno-stimulatory effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2011-806
Abstract 855: Flavokawain A inhibits urinary bladder cancer in UPII-mutant Ha-ras transgenic mouse bladder cancer model
Authors: Liu Zhongbo , Li Xuesen , Liu Shuman , Xu Xia , Sun Zheng , Simoneau Anne , Zi Xiaolin
Published: June 2012

Flavokawain A is the predominant chalcone identified from kava extract. We have recently shown that flavokawain A exhibited strong growth inhibitory effects on human bladder cancer cells both in vitro in cell cultures and in vivo in a xenograft mouse model. To examine whether flavokawain A affects specific-stages or course of bladder cancer development, the UPII-mutant Ha-ras transgenic mouse bladder model that mimics human papillary transitional cell carcinoma was used. The homozygous mutant Ha-ras transgenic mice were identified through genotyping using Southern blotting analysis and fed orally with 1g flavokawain A/kg food or 6 g flavokawain A /kg food for 3 or 6 months starting at 6 weeks of age. After treatments, organs are collected and weighted. H&E staining and immunohistochemstry were used to evaluate bladder cancer pathology and expression of biomarkers. All control food- fed male mice died of ureter obstruction and nephrohydrosis within 6 months of treatment, while more than 50% of mice fed with 6g flavokawain A /kg food for 6 months survived beyond the 6 months of treatment. The mean bladder weight in 6g flavokawain /kg food fed male mice also decreased by 66% compared to control treated mice for 3 months. Mouse pathology analysis showed that control mice exhibited poorly differentiated transitional cell carcinoma, while that flavokawain A treated mice presented well differentiated tumors. Compared to the dose of 6g/kg, a 1g/kg dose of flavokawain A is less effective in inhibition of bladder cancer development in this transgenic model. In addition, immunohistochemistry staining of Skp2 revealed that Skp2 oncoprotein expression in urothelium was also significantly decreased in flavorkawain A treatment group compared to control group. Taken together, our results suggest the potential usefulness of flavokawain A in treatment of superficial urinary bladder cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 855. doi:10.1158/1538-7445.AM2011-855
Abstract 86: DNA methylation of tumor suppressor and metastasis suppressor genes in circulating tumor cells
Authors: Chimonidou Maria , Strati Areti D. , Tzitzira Alexandra , Sotiropoulou Georgia , Malamos Nikos , Georgoulias Vassilis , Lianidou Evi
Published: June 2012

Background: Circulating tumor cells (CTCs) are associated with prognosis in a variety of human cancers and have been proposed as a liquid biopsy for follow up examinations. We show, that established tumor suppressor and metastasis suppressor genes (CST6, BRMS1, and SOX17) are epigenetically silenced in CTCs isolated from peripheral blood of breast cancer patients.

Methods: Peripheral blood was obtained from 56 patients with operable breast cancer, 27 patients with verified metastasis and 23 healthy individuals. Extraction of genomic DNA was performed in the EpCAM positive immunomagnetically selected CTC fraction of peripheral blood mononuclear cells (PBMC). DNA samples were subjected to sodium bisulphite conversion and tested for the presence of methylated and unmethylated gene promoter sequences by methylation specific PCR (MSP). All samples were also checked for CK-19 expression by RT-qPCR.

Results: In CTCs of patients with operable breast cancer, CST6 promoter methylation was observed in 17.9%, BRMS1 promoter methylation in 30.4%, and SOX17 promoter methylation in 53.6%. In CTCs of patients with verified metastasis, CST6 promoter methylation was observed in 37.0%, BRMS1 promoter methylation in 44.4%, and SOX17 promoter methylation in 74.1%.

Conclusions: Our results provide the first evidence that DNA methylation of tumor suppressor and metastasis suppressor genes is a hallmark feature of CTCs and confirm their heterogeneity. Our findings add a new dimension on the malignant nature of CTCs and may underlie the acquisition of malignant properties, including their stemlike phenotype. Moreover, these epigenetic markers open new possibilities for the exploitation of CTCs in monitoring cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 86. doi:10.1158/1538-7445.AM2011-86
Abstract 901: Specificities of CYFRA monoclonal antibodies KS 19.1 and BM 19.21 to cytokeratin 19
Authors: Vasko Ann-Jeanette , Thorne George D. , Kettlety Timothy R. , Li Zhong-Qian
Published: June 2012

CYFRA 21-1 measures cytokeratin fragments of cytokeratin 19 with the aid of two specific monoclonal antibodies (mAb), BM 19.21 and KS 19.1. CYFRA 21-1 has been adopted in several immunoassays as a tumor marker for non-small-cell lung cancer (Sanchez-Carbayo et al. Clin Chem 1999; 45: 1944-1953). We evaluated the binding of these antibodies to cytokeratin 19 and three other types of cytokeratin. Potential cross-reactivity of these antibodies with six cancer biomarker antigens was also evaluated. METHODS: SDS-PAGE and Western blotting were used to observe the binding of KS 19.1 and BM 19.21 with recombinant cytokeratin 8, 14, 18 and 19. An ARCHITECT CYFRA 21-1 assay was used to investigate whether cancer biomarker antigens AFP, beta-HCG, CA 125, CA 15-3, CA 19-9 and CEA interfere with the binding of these two antibodies to cytokeratin 19. 10 – 100 fold of the normal reference levels (8 ng/mL AFP, 5 mIU/mL beta-HCG, 35 U/mL CA 125, 37 U/mL CA 19-9 and 5 ng/mL CEA) of these biomarker antigens were spiked into five (5) serum samples. The concentration of the biomarker antigen CA 15-3 used for this study was 10 and 17 fold of its normal reference levels (31.3 U/mL). RESULTS: SDS-PAGE and Western blotting showed that KS 19.1 and BM 19.21 antibodies bind specifically to cytokeratin 19. KS 19.1 and BM 19.21 antibodies exhibited no binding to cytokeratin 8, 14 and 18 even though the total amount of cytokeratin 19 loaded was less than that of the other three cytokeratin's. Cancer biomarker antigens AFP, beta-HCG, CA 125, CA 15-3, CA 19-9 and CEA did not interfere with the binding of these two antibodies to cytokeratin 19 significantly. Mean cross reactivity of each biomarker was 5% (1% – 11%) for AFP, 4% (0% – 7%) for beta-HCG, 4% (0% – 15%) for CA 125, 6% (0% – 15%) for CA 15-3, 3% (0% – 6%) for CA 19-9 and 2% (0% – 4%) for CEA. CONCLUSIONS: Monoclonal antibodies KS 19.1 and BM 19.21 bound to cytokeratin 19 specifically and showed no cross reactivity with cytokeratin 8, 14 and 18 in Western blotting. Also cytokeratin 19-binding specificity was not significantly interfered significantly by cancer biomarker antigens AFP, beta-HCG, CA 125, CA 15-3, CA 19-9 and CEA in an ARCHITECT CYFRA 21-1 assay.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 901. doi:10.1158/1538-7445.AM2011-901
Abstract 952: Protein kinase C theta (PKCθ) and c-Jun regulate proliferation through cyclin D1 in KIT-independent gastrointestinal stromal tumors
Authors: Ou Wen-Bin , Fletcher Christopher D.M. , Demetri George D. , Fletcher Jonathan A.
Published: June 2012

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is standard of care for patients with metastatic GIST. However, most patients eventually develop clinical resistance to imatinib and other KIT/PDGFRA kinase inhibitors due to acquisition of secondary mutations in the KIT kinase domain, genomic amplification of KIT, or activation of alternate oncoproteins. Approximately 10% of KIT-dependent GIST metastases lose KIT expression at time of clinical progression during imatinib therapy. We performed whole transcriptome sequencing in KIT-expressing GIST vs. KIT-negative GIST, and thereby identified strong cyclin D1 expression only in KIT-negative GISTs. KIT-negative GISTs showed complete loss of the GIST biomarker, PKCθ, and restoration of PKCθ expression in these cells resulted in loss of Cyclin D1 expression. Lentivirus-mediated CCND1 (Cyclin D1) short hairpin RNA (shRNA) knockdown resulted in profound anti-proliferative and pro-apoptotic effects in KIT-negative GIST cell lines, and was associated with activation (dephosphorylation) of the tumor suppressor Rb and upregulation of the p27 CDK inhibitor. c-Jun expression levels paralleled the Cyclin D1 expression in the GIST lines, and c-Jun shRNA knockdown resulted in downregulation of cyclin D1 in the KIT-negative GIST lines, and was accompanied by profound anti-proliferative effects, including p27 upregulation, and pro-apoptotic effects as evidenced by PARP cleavage. By contrast, cyclin D1 and c-Jun silencing showed little anti-proliferative and pro-apoptotic effects in KIT-positive, KIT-dependent GIST lines. These findings show that cyclin D1 and c-Jun overexpression serve a crucial oncogenic role in KIT-independent GISTs, including those enabling clinical resistance to imatinib or sunitinib targeted kinase inhibitor therapies. Our findings also show that PKCθ downregulation and c-Jun upregulation coordinately enable cyclin D1 overexpression in KIT-independent GISTs. These novel findings highlight the relevance of c-Jun/cyclin D1 as novel therapeutic targets in GISTs that have lost KIT oncoprotein expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 952. doi:10.1158/1538-7445.AM2011-952
Abstract 972: Epigenetic-microRNA networks in colon cancer stem cells
Authors: Chatziapostolou Maria , Iliopoulos Dimitrios
Published: June 2012

Although chemotherapeutic regimens often suppress tumor growth, cancer patients show high variability in their responses and commonly develop resistance to these drugs and recurrence of the tumor. To explain this phenomenon, the cancer stem cell hypothesis suggests that tumors contain a small number of tumor-forming, self-renewing, cancer stem cells (CSCs) within a population of non-tumor-forming cancer cells. In our study, we have identified that CD133+/CD44+/ALDH1+ colon cancer cells are able to self-renew, form tumors in immunodeficient (rag2-/-gammac-/-) mice and are highly resistant to 5-fluorouracil (5-FU) chemotherapy treatment. Several studies have identified that microRNAs are central regulators of oncogenesis, however their role in CSC formation and maintenance has not been elucidated. In order to identify microRNAs that suppress the growth of CD133+/CD44+/ALDH1+ colon cancer stem HCT-116 cells, we transfected a library of 810 microRNAs in these cells and evaluated their viability 24h post transfection. The microRNA library screen revealed 8 microRNAs that suppressed more than 80% colon CSC growth. Specifically, miR-26b inhibited most effectively (96%) colon CSC growth. In addition, microRNA microarray analysis revealed that miR-26b expression is highly decreased in colon CSCs in comparison to colon non-stem cancer cells (NSCCs), suggesting the importance of miR-26b in colon CSC growth. Bioinformatic and molecular analyses revealed that miR-26b targets directly the 3’UTR of the enhancer of zeste homolog 2 (EZH2). Overexpression of EZH2 in colon NSCCs resulted in formation of colon CSCs, suggesting the importance of EZH2 in colon CSC formation. Furthermore, chromatin-immunoprecipitation-sequencing (ChIP-seq) analysis showed that miR-26b overexpression in colon CSCs reactivates the expression of genes repressed by H3K27 tri-methylation. Interestingly, we have identified that miR-26b promoter area is a direct target of EZH2 suppressed by H3K27 tri-methylation. The transcriptional signature of the miR-26b-EZH2 network was also identified in colon CSCs derived from human colon cancer tumors. In addition, miR-26b targeted selectively colon CSCs and acted together with 5-FU chemotherapy to block tumor growth and prolong remission in colon cancer mouse models. Taken together, these data suggest the identification of novel epigenetic-microRNA network in colon CSCs and provide the rationale and experimental basis for using the combination of miR-26b and chemotherapy drugs to improve treatment of patients with colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 972. doi:10.1158/1538-7445.AM2011-972
Abstract 984: The role of branched-chain amino acids (BCAAs) oxidation in the survival of brain metastatic breast cancer cells
Authors: Wu Xuefeng , Weihua Zhang
Published: June 2012

Background: Glucose is the main source of energy for most cells. When the glucose is limited some cells can use amino acids to support their survival. This ability of oxidizing branched chain amino acids (BCAAs) of the brain metastatic breast cancer cells was investigated.

Methods: Breast cancer cell lines, MDA-MB-231 cell and its brain metastatic derivative MDA-MB-Br3 were used in this study. The survivability, ability of oxidizing BCAAs and expression of BCAA oxidation related enzymes of these cells were determined.

Results: MDA-MB-Br3 cells survived well and oxidized more BCAAs than MDA-MB-231 cells when cultured in glucose free medium. Accordingly, the activity of branched-chain a-ketoacid dehydrogenase complex of MDA-MB-Br3 cells is significantly higher than that of the MDA-MB-231 cells.

Conclusions: The breast cancer cell line MDA-MB-Br3 can use BCAAs as a resource of energy for survival in the environment with very low level of glucose.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 984. doi:10.1158/1538-7445.AM2011-984
Abstract 991: Regulation of glucose metabolism by HIF in pancreatic cancer
Authors: Singh Pankaj K. , Purohit Vinee , Chaika Nina , Johnson Keith R. , Hollingsworth Michael A.
Published: June 2012

Pancreatic cancer is the fourth leading cause of cancer related deaths with a five-year survival rate of 5.5%. It is characterized by extremely aggressive tumor growth rate and high incidence of metastasis. Like other cancers, pancreatic cancer displays alterations in cellular metabolism, which holds significant promise for better management/cure of the disease. One of the most common and profound biochemical phenotypes of animal and human cancer cells is their ability to metabolize glucose at high rates, even under aerobic conditions. Several oncogenes and tumor suppressors have been implicated in altering tumor cell metabolism in order to facilitate tumor growth and metastasis. The objective of this study was to characterize the role of hypoxia inducible factors (HIF) in glucose metabolism in pancreatic cancer cell lines under normoxic and hypoxic conditions. We screened a panel of pancreatic cancer cell lines for glucose uptake under normoxic and hypoxic conditions. Although the effect of hypoxia on glucose uptake was variable in different pancreatic cancer cell lines, it correlated well with the expression levels of HIF-1alpha and HIF-2alpha. Furthermore, dual knockdown of HIF-1alpha and HIF-2alpha had similar negative effect on growth rate under hypoxic conditions, as did the treatment with glycolysis inhibitor 3-BrPA. These studies suggest that HIF factors regulate growth in pancreatic cancer by regulating glycolysis. Hence, combining strategies to target metabolism along with the strategies to target signal transduction might synergistically aid in diminishing cancer cell growth and invasiveness.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 991. doi:10.1158/1538-7445.AM2011-991