bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 731: Nuclear factor-κB activation and cetuximab-resistance in HNSCC cell lines
Authors: Hatakeyama Hiromitsu , Dahlman Kimberly Brown , Su Zengliu , Robbert Slebos , Pao William , Chung Christine H.
Published: June 2012

Background: Cetuximab, a monoclonal antibody targeting EGFR, has been shown to increase survival in patients with head and neck squamous cell carcinoma (HNSCC). However, only selective patients gain therapeutic benefit. To determine a mechanism of primary resistance to cetuximab, we characterized a primary keratinocyte cell line and 34 HNSCC cell lines for somatic mutations and gene expression alterations.

Experimental design: Thirty-five cell lines were assessed by 1) multiplex SNaPshot genotyping to detect 38 somatic mutations in the EGFR and its related pathways, 2) DNA sequencing of the tyrosine kinase domains of MET and FGFR3 to detect mutations, 3) qRT-PCR to quantify EGFR, MET and FGFR1 gene copy number (CN), 4) microarray analyses for gene expression profiles, and 5) matrigel colony formation assays to determine sensitivity to cetuximab. Gene mutations and CN changes were examined for potential association with cetuximab sensitivity. Differentially expressed genes between the cetuximab-resistant and -sensitive cell lines were assessed by GeneSpring and Ingenuity Pathways Analysis (IPA). To confirm the expression array data, seven representative cell lines were treated with BAY-11-7082, an IKB kinase inhibitor, to inhibit NF-kB activity.

Results: Three of 35 cell lines had mutations in PIK3CA (E542K, E545Q and H1047R). The cell line with E542K mutation was resistant to cetuximab while the cell lines with E545Q and H1047R were sensitive to cetuximab. No mutations were found in MET or FGFR3. CN gains were detected in EGFR and FGFR1 (20 cell lines with EGFR CN 4-10, 10 with EGFR CN >10, and 9 with FGFR1 CN 4-10), but not in MET. Interestingly, many of the cell lines with EGFR CN gains also had FGFR1 CN gains (Spearman rank test, r=0.48, p=0.0032). However, there was no correlation with the CN gains and cetuximab sensitivity. IPA showed genes involved in epithelial-to-mesenchymal transition (EMT) and NF-kB activation to be associated with cetuximab resistance. Cetuximab-resistant cell lines had higher expression of vimentin and lower expression of E-cadherin. BAY-11-7082 inhibited cellular proliferation of four cetuximab-resistant cell lines, but not three sensitive cell lines.

Conclusion: Mutations commonly seen in non-HNSCC cancers are rare, but EGFR and FGFR1 CN gains are common in HNSCC cell lines. The EMT phenotype and NF-kB activation may have an important role in cetuximab resistance and in novel therapeutic development in HNSCC.

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 731. doi:10.1158/1538-7445.AM2011-731
Abstract 745: HOXB7 renders breast cancer resistant to tamoxifen through activation of the HER2 pathway
Authors: Jin Kideok , Zhu Tao , Lundberg Goran , Sukumar Saraswati
Published: June 2012

Multiple factors, including long term treatment with tamoxifen, are involved in the development of selective estrogen receptor modulator (SERM) resistance of ERα positive breast cancer. It is well known in the endocrinology field that increased levels of ErbB/HER family members can also directly alter the cellular response to tamoxifen, but the mechanisms underlying increased expression of ErbB/HERs are not clear. In this studyreport we identifiedfound a number of HOXB7- specific binding sites throughout the genome in responsewith reaction to estrogen, through HOXB7 Chip on chip assay. We show that HOXB7 interacts with ERα and together they it bind to theharbors regulatory elements of ERα- regulated genes to enhance their transcription activity. Enforced expression of HOXB7 increases expression of ERα- regulated genes while knockdown of HOXB7 with siRNA reduces their expression. In addition, our work demonstrates that in order to mediate upregulation of HER2 in tamoxifen resistance, the HOXB7-ERα complex recruits ERα coactivators and promotes HER2 transcription activity through direct binding to the HER2 enhancer region. However, depletion of HOXB7 negatively affects the binding affinity of its cofactors to the HER2 enhancer element. Higher expression levels of HOXB7 significantly correlates with poorer disease free survival in ERα- positive breast cancer patients on adjuvant tamoxifen monotherapy. These findings implicate overexpression of HOXB7 as a key event in the initiation and maintenance of tamoxifen resistance. These studies suggest that HOXB7 acts as a key regulator orchestrating two major groups of target molecules, ERα and HER2, in the oncogene hieeirarchy. Therefore, fFunctional antagonism of HOXB7 has the potential tocould circumvent tamoxifen resistance.

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 745. doi:10.1158/1538-7445.AM2011-745
Abstract 747: Tumor vaccine adjuvant effect of TLR7 agonist Imiquimod is limited by inducing immusuppressive cells
Authors: Dang Yushe , Wagner Wolfgang M. , Gad Ekram , Disis Mary L.
Published: June 2012

Toll-like receptor (TLR) agonists are immune stimulators and have been studied as potential vaccine adjuvants with great interest. We evaluated the adjuvant effects of the TLR7 agonist imiquimod as compared to GM-CSF, which is widely used as a vaccine adjuvant in clinical trials. We found that both topical imiquimod and intradermal GM-CSF effectively induced the accumulation and activation of dentritic cells in draining lymph nodes and equally enhanced OVA specific CD4+ and CD8+ T cell responses in an OVA-tg mouse model. We further evaluated the efficacy of tumor prevention of imiquimod and GM-CSF in a neu-transgenic (neu-tg) mouse model of human breast cancer which over-expresses both neu and IGFBP2 tumor associated antigens. The neu-tg mice were immunized with a vaccine composed of three immunogenic IGFBP2 peptides prior to being inoculated with syngeneic MMC tumor cells. The tumor growth was significantly inhibited in the groups which received GM-CSF as an adjuvant, but not in imiquimod-treated groups. We questioned whether imiquimod induces immune suppression in response to tumor associated antigens and assessed the frequencies of myeloid-derived suppressive cells (MDSC) and T regulatory cells (Treg) in the splenocytes after three immunizations. We found that both CD11c+GR1+ MDSC and CD4+FOXP3+Treg cells were significantly increased in imiquimod-treated mice, but did not increase in GM-CSF-treated group. Serum levels of IL-10 were also significantly increased in imiquimod-treated, but not in GM-CSF-treated group. IFN-gamma secretion in response to IGFBP2 antigen, on the contrary, was remarkably enhanced in the GM-CSF-treated mice, but not in the imiquimod-treated mice as tested by an IFN-gamma ELISPOT assay. Thus, the TLR7 agonist imiquimod, as a vaccine adjuvant did not achieve a protective effect on breast cancer growth. The limited anti-tumor effect observed is associated with elevated levels of immunosuppressive MDSC and Treg cells. These results may have significant implications in the future development of imiquimod as a vaccine adjuvant.

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 747. doi:10.1158/1538-7445.AM2011-747
Abstract 748: TLR3 agonist Ampligen® as an adjuvant inhibits the growth of breast cancer
Authors: Dang Yushe , Gad Ekram , Lai Vy , Rastetter Lauren , Salazar Lupe L. , Nicodemus Christopher F. , Disis Mary L.
Published: June 2012

A potent adjuvant is the key for a successful vaccine against tumor antigens. We evaluated the adjuvant effect of a TLR3 agonist, Ampligen® on the growth of breast cancer in a transgenic mouse model. Ampligen® [rintatolimod; poly(I)·poly(C12,U)] is a synthetic mismatched double strand RNA that specifically stimulates TLR3 (Hemispherx Biopharma, Philadelphia, PA). A neu-tg mice [strain name, FVB/N-TgN (MMTVneu) 202Mul] over-expressing both neu and IGFBP-2 tumor associated antigens (Park KH et al CR 2008) were used for this study. Three IGFBP2 peptides were used as immunogen. The mice were vaccinated with the peptides and Ampligen® three times, 2 weeks apart, with PBS, peptides alone, and Ampligen® alone as controls. Syngeneic MMC tumor cells were injected into the mice two weeks after the last vaccine, and the growth of tumors was monitored afterwards. Three other immune modulators (GM-CSF, TLR7 agonist Imiquimod and TLR9 agonist CpG ODN 2395) were also used to evaluate the efficacy of Ampligen® and combination adjuvant approaches. Our results indicate that peptides alone did not mediate an anti-tumor effect. Although Ampligen® alone at high concentration (100ug) had a weak anti-tumor effect as compared to the control with 23% tumor inhibition, vaccination with IGFBP2 antigen and Ampligen® remarkably inhibited tumor growth by 60%. A dose response of Ampligen® was observed with 20ug as the optimal dose. The analysis of the levels of multiplex cytokines (IL-12p70, IL-1a, IL-1b, IL-2, IL-5, IL-10, IL-17, IFNg and TNFa) in serum two days after last vaccine showed that the level of IL-12p70 was increased in peptides/Ampligen® treated mice as compared to control (5.9±0.9 vs. 3.1±0.8 pg/ml;n=5; p=0.041) or other adjuvants. The serum levels of other cytokines were not statistically different. We compared the adjuvant effect of Ampligen® with GM-CSF, Imiquimod and ODN 2395. Besides Imiquimod, all these adjuvants significantly inhibited tumor growth as compared with peptides alone (p<0.002). Ampligen® was more effective than ODN2394 (p=0.02). The combination of Ampligen® with these other immunostimulators didn't further enhance the antitumor effect. In conclusion, Ampligen® as an adjuvant inhibited the growth of breast cancer with an increased level of Th1 cytokines and is a potent tumor vaccine adjuvant with the potential for clinical use.

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 748. doi:10.1158/1538-7445.AM2011-748
Abstract 749: Cancer stem cell vaccination confers significant anti-tumor immunity by selectively targeting cancer stem cells
Authors: Ning Ning , Teitz-Tennenbaum Seagal , Yet Ji , Li Mu , Moyer Jeffrey S. , Ginestier Christophe , Wicha Max S. , Xu Yingxin , Prince Mark EP , Chang Alfred E. , Li Qiao
Published: June 2012

To date, the majority of cancer stem cell (CSC) studies have been confined to human tumors inoculated into severely immunosuppressed hosts (e.g. SCID mice), where adaptive immune responses are absent, and such hosts are therefore not suitable for immunologic evaluation. The lack of immunocompetent syngeneic animal tumor models where stem cells can be isolated has been the primary obstacle to evaluating the immunogenicity of CSCs. In this study, we identified CSC-enriched populations in two histologically distinct murine tumors (melanoma B16-D5 and squamous cell cancer SCC7). We found that a very small percentage (0.5-5%) of ALDEFLUOR/ALDH1+ cells exist in these tumors; as few as 500 sorted ALDEFLUOR+ cells could form tumors in syngeneic hosts. The stem cell nature of the ALDELFUOR+ cells was confirmed by their ability to form spheres in culture and to self-renew in vivo. We evaluated the protective anti-tumor responses induced by D5 cancer stem cell-based vaccination with dendritic cell in the syngeneic immunocompetent host (B6 mice) in a pulmonary metastatic setting. We found that enriched D5 CSCs are immunogenic and are significantly more effective as an antigen source compared with unselected D5 tumor cells in inducing protective anti-tumor immunity. CSC-induced protective antitumor immunity was confirmed in a second tumor model (SCC7) syngeneic to a genetically different immunocompetent host (C3H) in a subcutaneous tumor setting. Immune sera collected from D5 or SCC7 CSC-vaccinated hosts contain high levels of IgG and IgG2b which bound specifically to D5 CSCs or SCC7 CSCs, respectively. This binding resulted in the CSC lysis in the presence of complement. In addition, CTLs generated from PBMCs harvested from D5 or SCC7 CSC-vaccinated hosts selectively killed D5 or SCC7 CSCs efficiently. We provide the first direct experimental evidence that selective targeting of cancer stem cells by CSC-primed antibodies and T cells may represent the mechanisms involved in CSC vaccine-conferred anti-tumor immunity. The findings from these studies may help develop novel cancer stem cell-targeted immunological approaches for cancer treatment.

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 749. doi:10.1158/1538-7445.AM2011-749
Abstract 760: Gene expression profiling in cancer vaccine: A new tool for cancer vaccine development
Authors: Matsueda Satoko , Sasada Tetsuro , Komatsu Nobukazu , Ioji Tetsuya , Shichijo Shigeki , Noguchi Masanori , Tashiro Kosuke , Kuhara Satoru , Itoh Kyogo
Published: June 2012

(Introduction) Recently, cancer vaccines have emerged as one of the promising therapeutic options. However, it is still difficult to predict clinical responses in vaccinated patients prior to vaccination. For further development of this field, it would be of critical importance to identify biomarkers for selecting patients adequate for cancer vaccination. To identify reliable biomarkers, we characterized gene expression profiles by the DNA microarray technology (Illumina) in PBMC from prostate cancer patients that showed better or worse responses to the personalized peptide vaccination (survival time > 900 or < 300 days, respectively; n = 20 each).

(Method) Total RNA was prepared from frozen-PBMCs prior to vaccination and hybridized. Detailed statistical analysis was performed to select genes that help predict patient clinical responses. In addition, to validate the result, 10 additional PBMC samples were analyzed in discriminant analysis.

(Result) A combination of genes was selected as a predictive biomarker. In discriminant analysis, the identified genes clearly discriminated long-term survivors from short-term survivors to the peptide vaccination.

(Conclusion) The identified gene set in PBMC may be useful as a biomarker in selecting patients adequate for personalized peptide vaccines. The detailed characterization of individual genes identified is currently underway.

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 760. doi:10.1158/1538-7445.AM2011-760
Abstract 781: Plasticity of human tumor-infiltrating Th17 cells
Authors: Ye Jian , Su Xinming , Hsueh Eddy C. , Zhang Yanping , Koenig Joyce M. , Hoft Daniel F. , Peng Guangyong
Published: June 2012

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases, their function in human tumor immunity remains largely unknown. We have recently identified Th17 cells as an important component of human tumor-infiltrating lymphocytes (TILs) obtained from melanoma, breast, colon and ovarian cancer patients, but their stability and plasticity in the tumor microenvironment is still unclear. In this study, we generated Th17 clones from TILs. We showed that Th17 clones can differentiate into IFN-gamma-producing and FoxP3+ cells after in vitro stimulation with OKT3 and allogeneic peripheral blood mononuclear cells (PBMCs). We further demonstrated that T cell receptor (TCR) engagement was responsible for this conversion, and that this differentiation was due to the epigenetic modification and reprogramming of gene expression profiles, including lineage-specific transcriptional factor and cytokine genes. In addition to expressing IFN-gamma and FoxP3, we showed that these differentiated Th17 clones mediated potent suppressive function after repetitive stimulation with OKT3, suggesting that these Th17 clones had differentiated into functional Treg cells. We further demonstrated that the Th17-derived Treg cells, unlike naturally occurring CD4+CD25+ Treg cells, did not reconvert back into Th17 cells even under Th17-biasing cytokine conditions. These results provide the critical evidence that human Th17 cells can differentiate into Treg cells and indicate the substantial developmental plasticity of Th17 cells, which may have clinical implications for the development of novel cancer immunotherapeutic approaches.

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 781. doi:10.1158/1538-7445.AM2011-781
Abstract 786: CEACAM1 regulates myeloid-cell-dependent tumor angiogenesis by inhibition of G-CSFR-Bv8 pathway
Authors: Lu Rongze , Shively John
Published: June 2012

Gr1+CD11b+ myeloid cells promote tumor angiogenesis and are refractory to VEGF antibodies treatment. Bv8, also known as Prokineticin 2, expressed in Gr1+CD11b+ cells and positively regulated by granulocyte colony-stimulating factor (G-CSF), has recently been shown to modulate Gr1+CD11b+ cells mobilization and tumor angiogenesis. Anti-Bv8 treatment reduced angiogenesis and growth. However, the signaling pathways in Gr1+CD11b+ to promote tumor angiogenesis are not fully understood. Carcinoembryonic antigen-related cell adhesion moclecule-1(CEACAM1) is highly expressed on Gr1+ myeloid cells, however the role of CEACAM1 in myeloid cells mediated tumor angiogenesis has not been investigated.

We have demonstrated that tumor growth and angiogenesis was significantly enhanced in Ceacam1−/− mice, which was independent on T and B cells by backcrossing Ceacam1−/− mice with Rag1−/− mice. Meanwhile Gr1+CD11b+ cells infiltration in tumor, spleen and blood was increased. Tumors coinjected with Gr1+CD11b+ cells from Ceacam1−/− mice exhibited increased angiogenesis compared to coinjection with Gr1+CD11b+ cells from control mice. Depletion of Gr1+ cells inhibited tumor growth and angiogenesis in control and Ceacam1−/− mice.

Gr1+CD11b+ cells from Ceacam1−/− mice expressed much higher Bv8 in vitro and in vivo. Further we have shown that CEACAM1 recruited Src-homology-phosphatase-1 (SHP-1) to attenuate granulocyte colony-stimulating factor receptor (G-CSFR)-Bv8 pathway in myeloid cells. Reconsititution of CEACAM1 into Ceacam1−/− bone marrow cells restored tumor growth and angiogenesis to normal level, and mutations of immunoreceptor tyrosine-based inhibitory motifs ITIMs abolished this restoration. Therefore, we identified CEACAM1, as an inhibitory regulator of G-CSFR-Bv8 pathway, negatively regulates myeloid cells dependent tumor angiogenesis.

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 786. doi:10.1158/1538-7445.AM2011-786
Abstract 789: Cytokine interleukin-16 is an important growth-promoting factor for multiple myeloma
Authors: Atanackovic Djordje , Hildebrandt York , Reinhard Henrike , Templin Julia , Bokemeyer Carsten , Kröger Nicolaus
Published: June 2012

BACKGROUND: Multiple myeloma is a malignancy characterized by the expansion of a plasma cell clone that localizes to the human bone marrow. Myeloma cells and bone marrow stroma cells both produce soluble factors promoting the survival and progression of multiple myeloma. Interleukin-(IL)-16 is involved in regulating migration and proliferation of normal leukocytes, however, it has been unclear whether IL-16 also plays a role in the pathophysiology of human cancers.

METHODS: Using an antibody array we screened BM plasma of MM patients for the presence of a large number of cytokines/chemokines. We confirmed cytokine expression by myeloma cell lines and malignant plasma cells from MM patients applying real-time PCR, western blots, ELISA, and flow cytometry. We used transfection with inhibitory RNA to analyze cytokine function and we applied anti-cytokine antibodies to evaluate possible therapeutic options for MM.

RESULTS: We found IL-16 to be strongly overexpressed in the bone marrow of myeloma patients. Myeloma cell lines as well as primary tumor cells from myeloma patients constitutively expressed IL-16 and its receptors CD4 and/or CD9 and spontaneously secreted soluble IL-16. Silencing of IL-16 had an anti-proliferative effect on the tumor cells which was reversed by the addition of a recombinant C-terminal IL-16 peptide. Most importantly, the application of a monoclonal antibody directed against IL-16 had a strong growth-inhibiting influence on myeloma cells.

CONCLUSIONS: These findings suggest that cytokine IL-16 is an important growth-promoting factor in MM and might represent an important diagnostic and therapeutic target for this incurable human malignancy.

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 789. doi:10.1158/1538-7445.AM2011-789
Abstract 792: Tumor-induced thymic atrophy: Alteration in interferons and Jak/Stats signaling pathways
Authors: Lopez Diana M. , Carrio-Jardines Roberto , Torroella-Kouri Marta , Iragavarapu-Charyulu Vijaya
Published: June 2012

The thymus is the major site of T cell differentiation and a key organ of the immune system. Most important, it provides a specialized environment for the selection of rearranged clones that will function appropriately in the adaptive immune response. Thymic involution has been observed in several model systems; including graft-vs-host disease, aging, and tumor development, however, the mechanisms involved in this phenomenon remain to be elucidated. Previous results from our laboratory have reported that the severe thymic atrophy and impaired T cell development seen in mammary tumor bearers are associated with an arrest in at least two steps of T cell differentiation, changes in the levels of crucial cytokines expressed in the thymus microenvironment, and a progressive increase in apoptosis during the tumor development mainly due to downregulation of important molecules that control programmed cell death. Cytokines regulate numerous aspects of hematopoiesis via activation of the Jak/Stat pathways. In the present study we have used our mammary tumor model to investigate whether changes in the levels of cytokines in the thymus could affect the normal expression of the aforementioned pathways. RNA and protein analysis revealed an overexpression of the different interferons, a downregulation of most of the Jak/Stat pathways, and an increased expression of several suppressors of cytokine signaling (SOCS) in the thymuses of tumor bearers. Collectively, our data suggest a mechanism by which the impaired Jak/Stat signaling pathways observed in the whole thymus of tumor-bearing mice could be contributing to the abnormal T cell development and apoptosis observed during the tumor-induced thymic atrophy.

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 792. doi:10.1158/1538-7445.AM2011-792
Abstract 798: Measuring TK1 autoantibodies in the serum of cancer and non-cancer patients
Authors: LaPorte Wesley , Abegg-Lawrence Taylor , Alegre Melissa M. , Robison Richard A. , O'Neill Kim L.
Published: June 2012

One of the foremost problems in cancer is lack of a convenient, early detection method. Often malignancies are discovered too late, at stages that are so evolved that treatment becomes painful, expensive, or ineffective. Thymidine Kinase 1 (TK1) is an important nucleotide salvage pathway enzyme involved in DNA synthesis and repair. It has been shown that TK1 levels increase under normal cellular conditions typically during cell proliferation. However under malignant conditions, TK1 is over-expressed both in the cytosol of cancer cells and the serum of cancer patients. Consequently, serum TK1 levels (sTK1) are often used in cancer diagnosis and prognosis. We hypothesized that there may be autoantibodies against sTK1 in cancer patients because TK1 is generally intercellular and usually sequestered from the immune system. In order to investigate this hypothesis, we developed an ELISA in a 96 well plate format specifically to detect and measure the presence of autoantibodies against sTK1. Using purified TK1 as a capture antigen, we added serum from both pre-treatment cancer patients and age matched normal patients to detect any autoantibodies that interact with sTK1. Using two different types of primary, HRP conjugated antibodies, (Goat Anti-Human-HL & Goat Anti-Human-Fc) we were able to determine that there are antibodies to TK1 in the serum of both cancer and non-cancer patients. We eliminated the possibility that we were only detecting fragments of autoantibodies that were not true autoantibodies since our GAH-Fc antibody is specific to only intact antibodies. Furthermore, when we added TCEP (A common reducing agent which we used to remove the antibodies from the serum) to the serum, we saw a measurable signal reduction. Preliminary data suggest there are autoantibodies to TK1 in both cancer serum and normal serum. Further research is underway to elucidate the mechanisms responsible.

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 798. doi:10.1158/1538-7445.AM2011-798
Abstract 8: Identification of a non-canonical BH3 peptide that binds the BH3 pocket of Mcl-1
Authors: Placzek William J. , Sturlese Mattia , Wei Jun , Wu Bainan , Cellitti Jason , Pellecchia Maurizio
Published: June 2012

The identification of selective inhibitors of the anti-apoptotic Bcl-2 family remains a principal target for cancer drug development. With the recent recognition of Mcl-1 as the primary Bcl-2 family member expressed in solid tumors and its ability to enable therapeutic resistance, identification of peptides that specifically bind to Mcl-1 has become critical for the development of improved therapeutics. To identify novel peptides that bind to the BH3 cleft of Mcl-1, we screened a randomized twelve residue phage display library using a pan-active Bcl-2 small-molecule inhibitor. This screen identified a unique non-canonical BH3 sequence, rBH3, which selectively binds to Mcl-1 over Bcl-xL. NMR analysis of the rBH3 interaction with Mcl-1 shows that it binds to the BH3 binding groove and an alanine scan of the sequence highlights the key residues responsible for its binding. A search for proteins with the rBH3 motif has identified a number of interesting targets which have previously been associated with apoptosis regulation. Thus we believe that this sequence represents a unique, Mcl-1-specific peptide sequence that may identify a previously unknown family of Mcl-1 interacting proteins.

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 8. doi:10.1158/1538-7445.AM2011-8
Abstract 807: Hydrogen sulfide-releasing aspirin modulates xenobiotic metabolizing enzymes in vitro and in vivo
Authors: Street Cherease R. , Chattopadhyay Mitali , Kodela Ravinder , Boring Daniel , Crowell James A. , Kashfi Khosrow
Published: June 2012

Introduction: Xenobiotic metabolizing enzymes are classified as phase-I (oxidation/reduction) and phase-II (conjugation) enzymes; the balance between the phase-I carcinogen-activating enzymes and the phase-II detoxifying enzymes may be important in determining an individual's risk for cancer. Hydrogen sulfide-releasing aspirin (HS-ASA) is a new compound with significant anti-inflammatory properties. It consists of ASA covalently attached to 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, which releases hydrogen sulfide. Our initial studies indicate that HS-ASA has chemopreventive properties. In the present study, we evaluated the effect of HS-ASA on xenobiotic metabolizing enzymes both in vitro and vivo.

Methods: HS-ASA was synthesized and purified by us with 1H-NMR verification. Cell lines: HT-29 human colon adenocarcinoma and Hepa 1c1c7 mouse liver adenocarcinoma. Cell growth inhibition: MTT. Levels of cytochrome P450 1A1 (CYP 1A1) a phase-1 enzyme and NAD(P)H:quinone oxireductase (NQO), glutathione S-transferase (GST) and UDP-glucuronyltransferase (UGT), phase-II enzymes by immunoblots. Enzyme activity: GST and thioredoxin reductase (Trx). In vivo: Male Wistar rats (N = 4/group) were treated via gavage either with vehicle or with HS-ASA 100 mg/kg/day. Weight of animals in each group was recorded every 3 days. After 21 days, animals were killed and liver microsomes and cytosols were prepared for phase-I and phase-II enzyme evaluation.

Results: HS-ASA inhibited the growth of HT-29 and Hepa 1c1c7 cells, with an IC50 of 2.5 ± 0.3 µM and 2.9 ± 0.4 µM, respectively. The IC50 for ASA in both cell lines was >5,000 µM at 24h. The ratio ASA/HS-ASA is >2000 in the HT-29 cells and >1700 in Hepa 1c1c7 cells, suggesting that HS-ASA is at least 1700-2000-fold more potent than traditional ASA. In HT-29 cells, at 0.5xIC50, 1xIC50, and 2xIC50, HS-ASA increased GST activity by 2.5-, 7.2-, and 11-fold; and it inhibited Txr by 36 ± 3%, 67 ± 2% and 79 ± 2% respectively. In HT-29 cells, levels of CYP 1A1 were not significantly affected whereas levels of GST and UGT were increased dose-dependently by HS-ASA. In Hepa 1c1c7 cells, levels of NQO were also significantly increased. In vivo, HS-ASA: 1. had no effect on rat's weight; 2. increased hepatic GST activity by 3.4-fold and NQO by 1.4-fold; 3. immunoblots showed significant increases in GST and NQO protein levels but no changes in CYP 1A1 protein levels.

Conclusions: HS-ASA co-induces multiple phase-II metabolizing enzymes and thus may be regarded as a monofunctional inducer. This represents one mechanism by which HS-ASA exhibits chemopreventive properties.

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 807. doi:10.1158/1538-7445.AM2011-807
Abstract 813: Deletion of vitamin D receptor in prostate epithelial cells increases proliferation, reduces apoptosis, and enhances early carcinogenesis in the TgAPT121 mouse model of prostate cancer
Authors: Kovalenko Pavlo L. , Clinton Steven K. , Fleet James C.
Published: June 2012

High vitamin D status has been proposed to prevent prostate cancer (PCa). In vitro, the active metabolite of vitamin D suppresses PCa cell growth and has a number of other anti-cancer actions in prostate epithelial cells (PEC) that are mediated through a vitamin D receptor (VDR)-dependent transcriptional mechanism. We examined the in vivo importance of VDR in normal PEC biology and in PCa development using mouse models. In the first study, 7 wk-old wild type mice and mice with PEC-specific deletion of VDR (PEC-VDR KO) were randomized to intact, castration, or castration + testosterone propionate (TP, 2.5 mg/kg BW for last 5 d) groups and mice were killed 12 d later. PEC proliferation (Ki-67) and apoptosis (TUNEL) were determined in the anterior prostate (AP). Castration reduced proliferation and increased apoptosis indices in the prostate while TP treatment reversed these effects. In PEC-VDR KO mice, TUNEL staining was significantly reduced (e.g. 50% lower after castration) and Ki-67 staining was significantly increased (e.g. 2-fold higher after castration + TP) compared to the controls. This suggests that loss of VDR signaling provides an environment in the prostate that may be permissive to events that enhance prostate carcinogenesis. We tested this hypothesis in the TgAPT121 transgenic mouse model of PCa. The TgAPT121 transgenic mice express a truncated SV40 Large T antigen (T121) in PEC. T121 expression inactivates the retinoblastoma (Rb) family of proteins causing epithelial cell hyperplasia, development of prostatic intraepithelial neoplasia (PIN) lesions by 12 wk of age, and microinvasive adenocarcinoma by 6 mo of age. TgAPT121 (APT;VDR WT) mice and TgAPT121 lacking both VDR alleles in PEC (APT;PEC-VDR KO) were generated and at 28 wks of age their prostates were removed and histologically graded. The total area covered by high grade PIN, adenocarcinoma, and microinvasive cancer was significantly higher in APT;PEC-VDR KO as was the number of foci per AP for each of these lesions (e.g. adenocarcinoma lesion foci 7.93% in APT;PEC-VDR KO versus 2.55% in APT;VDR WT, p<0.01). PEC VDR deletion significantly increased the histological grade of carcinogenic lesions in APT;PEC-VDR KO (score = 17.47 vs. Control = 16.32, p<0.05). This demonstrates that VDR signaling is critical for the anticancer effects of vitamin D and that signaling through the VDR delays transition of normal epithelium to PIN and early carcinoma.

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 813. doi:10.1158/1538-7445.AM2011-813
Abstract 817: Isothiocyanates inhibit proteasome activity and proliferation of multiple myeloma cells
Authors: Mi Lixin , Gan Nanqin , Chung Fung-Lung
Published: June 2012

Isothiocyanates (ITCs), including benzyl ITC (BITC), phenethyl ITC (PEITC), and sulforaphane, induce cell cycle arrest and apoptosis in a variety of cancer cells and animal models. Although studies show that reactive oxygen species (ROS) generation may be responsible, at least in part, for apoptosis induced by ITCs, our recent studies show that covalent binding to target proteins may actually be an important event triggering apoptosis (Cancer Res., 2007, JBC.,2008, and JBC., 2009). In this study, we report that BITC and PEITC significantly inhibit proteasome activity in a variety of cell types. Further studies show that ITCs inhibit both the 26S and 20S proteasome, presumably through direct binding, and that this inhibition is unrelated to either ROS generation or ITC-induced protein aggregation. The potency of ITC-induced proteasome inhibition correlates with the rapid accumulation of p53 (tumor suppressor) and IκB (NF-κB inhibitor). Finally, our results demonstrate that BITC and PEITC as stronger proteasome inhibitors significantly suppress growth of multiple myeloma cells through induction of cell cycle arrest at G2/M phase and apoptosis. These results suggest that proteasome, like tubulin, is a potential molecular target of ITCs, unraveling a novel mechanism by which ITCs may inhibit multiple myeloma cell growth. This study provides new leads for ITC compounds with therapeutic and preventative efficacies for multiple myeloma.

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 817. doi:10.1158/1538-7445.AM2011-817
Abstract 826: Potential Combination Therapy to Combat Endometrial Cancer
Authors: Daikoku Takiko , Jackson Lindsey , Dey Sudhansu K.
Published: June 2012

Endometrial cancer (EMC) is the most common gynecological malignancy causing approximately 7,000 deaths each year in the United States. The underlying causes of EMC are not clearly understood, and treatment options for patients with advanced stages are limited. The gene with the highest frequency of alteration in EMC is Phosphatase and tensin homolog (Pten). The loss of Pten results in increased PI3K activity with enhanced Akt activation. Increased levels of activated Akt (pAkt) stimulate both cyclooxygenase-2 (COX-2) and mammalian targets of rapamycin complex 1 (mTORC1) activity. Although these activities are also heightened in human EMC, it is not clear whether they are the critical players in cancer initiation and progression or whether they work interactively. Animal models that spontaneously develop cancers are powerful tools for studying mechanisms of cancer initiation and progression and for developing treatment strategies. We recently generated an EMC mouse model in which uterine Pten was conditionally deleted by crossing floxed Pten mice with progesterone receptor (PR)-Cre (PRcre/+) mice. All PtenloxP/loxP/PRcre/+ mice developed EMC by one month of age with invasion occurring by 3 months. Tumor phenotypes in these mice mimic many aspects of human EMC. Using this mouse model, we found that uteri of these mice show heightened levels of COX-2 and mTORC1 activities. These observations led us to examine whether inhibiting COX-2 and/or mTORC1 activities reduces the growth of EMC. PtenloxP/loxP/PR+/+ (control) and PtenloxP/loxP/PRcre/+ mice were treated with rapamycin (an inhibitor of mTORC1 signaling) and/or celecoxib (an inhibitor of COX-2 activity) by oral gavages every alternate day starting from 30 days of age for 29 days. We found that although treatment with rapamycin or celecoxib attenuated tumor growth, maximum reduction in tumor growth and progression was noted for PtenloxP/loxP/PRcre/+ females receiving both rapamycin and celecoxib. These results suggest that a combined treatment with celecoxib and rapamycin is an effective therapeutic strategy for combating EMC. This study was supported in parts by grants from NIH-P01-CA-77839 (S.K.D), Concern Foundation (T.D.) and Ohio Cancer Research Associates (T.D.).

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 826. doi:10.1158/1538-7445.AM2011-826
Abstract 835: Elevated in utero estrogenic environment may increase later breast cancer risk by down-regulating miRNAs
Authors: Hilakivi-Clarke Leena A. , de Assis Sonia , Clarke Robert , Warri Anni , Marian Catalin , Zwart Alan , Jin Lu , Kim Dominic , Tian Ye , Zhang Bai , Wang Yue , Xuan Jason Jianhua
Published: June 2012

Objectives: High maternal estrogen levels during pregnancy increase a female offspring's breast cancer risk in women and in animal models. This increase is proposed to be mediated by epigenetic changes that involve DNA methylation. Dynamic changes in miRNA expression during fetal development also play a key role in setting gene transcription patterns that persist through the life-time. In this study, we investigated whether in utero exposure to excess estradiol (E2) causes persistent changes in the expression of miRNAs and their target genes. Methods: Pregnant Sprague Dawley rats were fed AIN93G diet supplemented with 0.1 ppm ethinyl estradiol between gestation days 13 and 20. Offspring's mammary glands were obtained on postnatal day 50, and miRNAs were measured using applied Biosystems TaqManR Array Rodent MicroRNA and mRNAs using RG_U34A Affymetrix platform. Data generated in the two arrays were analyzed by an integrated approach which uses a sequence-based TargetScan method to predict miRNA targets and then correlates these targets with mRNA data to identify targets specific to in utero E2 exposure. Results: Maternal E2 exposure during pregnancy increases carcinogen-induced mammary tumorigenesis in the offspring (final tumor incidence in controls: 56%, in E2 offspring: 82%, p<0.05). Fifteen miRNAs are significantly down-regulated in the mammary glands of adult rats exposed to E2 in utero, compared to the controls. Many of these are known to be down-regulated by E2 in MCF-7 human breast cancer cells including miR-21, miR-23ab, miR-27ab, and miR-200c. Some of these miRNAs and others that are down-regulated in the mammary glands of in utero E2 exposed rats (let-7, miR-26ab, miR-181, and miR-203), in turn, suppress the expression of estradiol-regulated genes, as defined by Cicatiello et al., 2010. We also found that of the known ER-α activated genes, more than 10% are significantly up-regulated in the E2 group. These genes included BCL2L1, CYP1B1, and IGF1R. Conclusions: Our findings indicate that maternal exposure to excess E2 during pregnancy may cause a persistent up-regulation of estrogen-regulated genes by suppressing miRNAs that target these genes. Of note is that some of the miRNAs found to be down-regulated in the E2 group suppress genes known to be involved in DNA methylation. For example, miR-21 targets DNMT1; our study indicates that mammary glands of rats exposed to excess E2 in utero express significantly elevated DNMT1 levels, compared to the controls. Thus, changes in miRNAs may also be involved in inducing DNA methylation.

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 835. doi:10.1158/1538-7445.AM2011-835
Abstract 837: Novel sulindac derivatives that inhibit colon cancer growth by a COX independent mechanism involving PDE5 inhibition and the suppression of nuclear β-catenin levels
Authors: Whitt Jason D. , Keeton Adam B. , Tinsley Heather N. , Gary Bernard D. , Li Nan , Mathew Bini , Reynolds Robert C. , Piazza Gary A.
Published: June 2012

Many colon cancers harbor APC mutations that prevent the proteosomal degradation of oncogenic β-catenin. Previous studies have found that certain nonsteroidal anti-inflammatory drugs (NSAIDs) have the ability to reduce nuclear levels of β-catenin and inhibit tumor cell growth through a cyclooxygenase (COX)-independent mechanism, which might be responsible for their cancer chemopreventive activity. A potential mechanism may involve cGMP phosphodiesterase (PDE) inhibition and the activation of protein kinase G (PKG). In this study we evaluated over 500 novel sulindac derivatives for colon tumor cell growth inhibitory activity. Select compounds were also evaluated for COX and cGMP PDE inhibitory activity to determine which activity is associated with colon tumor cell growth inhibition and ability to suppress nuclear β-catenin levels. A subset of derivatives was identified that displayed reduced COX-1 and COX-2 inhibitory activity, yet increased potency to inhibit the growth of several colon tumor cell lines (HT29, HCT116, SW480, and COLO741) with IC50 values of 2-10 µM compared with IC50 values of 80-110 µM for the parent compound, sulindac sulfide (SS). Certain derivatives also displayed increased potency to inhibit recombinant phosphodiesterase 5 (PDE5), which appears to be the isozyme responsible for the cGMP PDE inhibitory activity of SS. Treatment of colon tumor cells with such compounds also increased the phosphorylation of vasoactive stimulated protein (VASP), a known PKG substrate. Furthermore, nuclear levels of β-catenin decreased within the same time period as PKG activation, which is consistent with previous reports suggesting that PKG can phosphorylate β-catenin to induce proteosomal degradation. Together, these data indicate that sulindac can be chemically modified to eliminate COX inhibitory activity while enhancing its ability to inhibit colon tumor cell growth. We conclude that the suppression of nuclear β-catenin levels by SS is independent of COX inhibition and that novel derivatives can be developed with improved antineoplastic activity while reducing the toxicities associated with COX inhibition. Funding provided by NIH/NCI grants CA131378 and CA148817.

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 837. doi:10.1158/1538-7445.AM2011-837
Abstract 841: Inhibition of head and neck squamous cell carcinoma growth by combination treatment of erlotinib and celecoxib: Potential chemopreventive agents
Authors: Zhang Hongzheng , Nannapaneni Sreenivas , Chen Zhengjia , Saba Nabil , Khuri Fadlo R. , Chen Georgia (Zhuo) , Shin Dong M.
Published: June 2012

The interaction of epithelial growth factor receptor (EGFR) and cyclooxygenase 2 (COX-2) pathways plays an important role in tumorigenesis. Our recent Phase I chemoprevention trial of premalignant lesions including pathologically moderate or severe dysplasia and carcinoma in situ in oral cavity and larynx showed a 71% (5/7) estimated overall response rate after a 6-month-long combination treatment of Erlotinib and Celecoxib without significant toxicities (ASCO 2010 Abstract # 5535). The current preclinical study was designed to investigate the underlying mechanisms that contribute to the inhibitory effect on the disease progression by the combined treatment of Erlotinib and Celecoxib. Chemopreventive efficacy was determined using a head neck squamous cell carcinoma (HNSCC) xenograft model with Tu212 cell line. Animals received vehicle control (0.1% Tween 80 and 0.5% methylcellulose, n=6), Celecoxib at 50mg/kg body weight (n=6), Erlotinib at 75mg/kg body weight (n=7), or combination of Erlotinib and Celecoxib (n=8) with the same dose as the single agent by oral gavaging daily for 6 days before subcutaneous implantation of cancer cells. Animals were further treated daily for 5 days per week with a total of 4 weeks. The combination treatment significantly suppressed tumor growth compared with Erlotinib alone (p=0.01), Celecoxib alone (p<0.0001) or the vehicle control (p<0.0001). In vitro study was performed with Tu212 and Tu686 HNSCC cell lines. Cell cycle analysis revealed that G1 arrest was induced by either Erlotinib alone (59% and 67% for Tu212 and Tu686, respectively), or Celecoxib alone (50% and 58% for Tu212 and Tu686 cells, respectively). The combination treatment resulted in greater G1 arrest than either single agent alone (72% in Tu212 and 65% in Tu686 cells). Molecular analysis showed that the combined treatment augmented suppression of pERK, pAKT and pEGFR. In addition, Erlotinib suppressed mTOR pathway by diminishing pS6. These results support the findings in the phase I study. The study has progressed to Phase II clinical trial. In conclusion, augmented inhibition of pEGFR/pERK/pS6 pathways could contribute to the synergism by Erlotinib and Celecoxib in suppressing HNSCC tumor growth. (This study is supported by NIH grant U01 CA151802 and P50CA128613 to Dr. Shin and Georgia Cancer Coalition Distinguished Cancer Scholar award to Drs Shin and Chen. *: These authors had equal contribution.)

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 841. doi:10.1158/1538-7445.AM2011-841