bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2636: Mechanisms of therapy-induced senescence with ABT-737
Authors: Song Jin H. , Kraft Andrew S.
Published: June 2012

Aberrant expression of the antiapoptotic Bcl-2 family of proteins in human cancer is correlated with poor outcomes after standard chemotherapy. ABT-737 is a cell permeable Bcl-2 family antagonist that is capable of binding to Bcl-2, Bcl-xL, and Bcl-w. ABT-737 functions by displacing BH3 proteins, such as Bim, from these proteins activating Bax and Bak to induce apoptotic cell death. Resistance to ABT-737 appears to be mediated by the elevated expression of Mcl-1 or Bfl-1/A1 which are not capable of binding this compound. Since renal, prostate and lung carcinomas, exhibit elevated levels of endogenous Mcl-1 many of these tumor cell lines are resistant to killing by this agent.

Gene chip analysis in order to understand anticancer action of ABT-737 in human cancer cells reveals an intrigue data that ABT-737 regulates key genes involving cytokines and chemokines associated with senescence phenotype. Because senescence is considered to be a key mechanism that protects against cancer development, we investigated the mechanism of action for ABT-737 in human cancer cells where this compound is incapable of inducing apoptotic cell death, inhibit growth of tumor cells by the induction of cell senescence. Here, we identify a novel pathway involving the induction of DNA damage response by caspase-3 cleavage that normally induces apoptosis, which instead leads to promotion of cellular senescence.

Treatment of renal, lung and prostate cancer cell lines with ABT-737, but not an inactive enantiomer, caused the induction of senescence-associated β-galactosidase, and additional changes associated with the senescence phenotype including increases in specific gene transcription, secretion of IL-6 and IL-8, and stimulation of the activity of C/EBPβ. ABT-737 treatment of these cell lines induced activation of the DNA damage response pathway as demonstrated by increases in γ-H2AX and phosphorylation of both the ATM and Chk2 protein kinases. Decreasing ATM levels with shRNA blocked the senescent phenotype suggesting that this pathway was critical for the induction of ABT-737-mediated senescence. DNA damage induced by ABT-737 treatment was caused by low level activation of the caspase cascade, which was blocked by decreasing caspase-3 levels with shRNA. ABT-737-induced senescence appeared to be p53-dependent, since the overexpression of dominant-negative p53 inhibited the ABT-737-induced increases in p21 protein levels, β-galactosidase, and IL-6 transcription.

These findings suggest that in multiple cancer cell types where ABT-737 is incapable of inducing sufficient DNA damage to activate apoptosis, it instead inhibits cancer cell growth by inducing senescence, a phenotype that is associated with major changes in gene transcription and protein secretion.

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 2636. doi:10.1158/1538-7445.AM2011-2636
Abstract 2646: N-ethylmaleimide modulates gemcitabine effect on ribonucleotide reductase M1 subunit (RRM1)
Authors: Chen Zhengming , Bepler Gerold
Published: June 2012

Ribonucleotide Reductase M1 (RRM1) is the regulatory subunit of the holoenzyme that catalyzes the conversion of ribonucleotides (NTP) to 2’-deoxyribonucleotides (dNTP) in mammalian cells. Its function is indispensible in cell proliferation and DNA repair. It is also a crucial marker of therapeutic efficacy in various cancers. This study investigated how the alkylating agent N-ethylmaleimide (NEM) interacts with the antimetabolite gemcitabine in inhibiting its target protein RRM1.

Lung cancer cell lines and the human embryonic kidney cell line (HEK293) were used for transfection, immunoprecipitation, and proliferation assays. Site-directed mutagenesis was used to create point mutations. The protein ID and modification sites were identified by LC/MS/MS. Flow cytometry was used for sorting and plating cells. Mouse xenografts were used for in vivo validation.

When cells were treated with gemcitabine and the alkylating agent NEM, a novel 110kD band, along with the 90kD native RRM1 band, was recognized in immunoblots. This 110kD band was identified as RRM1 by LC/MS/MS. It is specific to gemcitabine/NEM, among 11 chemotherapy drugs and 3 alkylators tested. It can also be detected in the mouse xenografts treated with gemcitabine. We mutated 7 residues that are essential for the RRM1 activity to alanine. Among these mutants, C218A, C429A, and E431A did not show any 110kD band, while C444A, N427A, C787A and C790A showed diminished 110kD, suggesting the 110kD RRM1 is involved in functionality. This was further confirmed by proliferation assays with gemcitabine and NEM in wild type and mutant cells.

In conclusion, the alkylator NEM specifically modulates the gemcitabine-RRM1 interaction and understanding the mechanism may lead to improved efficacy of and reduced resistance to gemcitabine.

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 2646. doi:10.1158/1538-7445.AM2011-2646
Abstract 2683: Identification of tumor antigens in neu transgenic mice as diagnostic targets for the early detection of human breast cancer
Authors: Mao Jianning , Ladd Jon , Wu Mei , Gad Ekram , Lu Hailing , Hanash Samir , Disis Mary
Published: June 2012

Tumor antigens, expressed early in disease, which can induce specific antibody response could serve as biomarkers for early detection of human breast cancer. However, the ability to obtain sera from women prior to the development of breast cancer is limited, which makes it difficult to identify early antigens directly from breast cancer patients. Transgenic mouse models have been proved to demonstrate similar antigen repertoire to breast cancer patients. Therefore, we propose to identify early tumor antigens in neu transgenic mice and use them as diagnostic targets for early detection of human breast cancer. We have collected serial sera from neu transgenic mice, sera collected before the animals develop palpable tumor “pre-diagnostic sera”. With serological analysis of recombinant cDNA expression libraries (SEREX) approach, we were able to identify nine tumor antigens from pre-diagnostic sera, Rpl5, TNFaip3/A20, Pdhx, Otud6b, Stk39, Zfp238, Dnajc10, Lgals8 and Vps35. These antigens demonstrated detectable IgG antibody response prior to tumor development in neu transgenic mice. IgM is the first antibody to be produced during a humoral immune response, so we questioned whether including antigen specific IgM with IgG responses could achieve a better diagnostic performance. Firstly, we analyzed receiver-operating-characteristic (ROC) curves of these antigens by IgG and IgM ELISA analysis in neu transgenic mice and 26 FVB control mice. By combining IgG and IgM ELISA, we were able to achieve a higher AUC: Otud6b and Stk39 show an AUC of 0.882, and adding Lgals8 IgG ELISA to this panel can achieve an AUC of 0.924 in discriminating cases from control mice. We documented that these autoantibodies are also detectable in the serum of women with breast cancer and then screened a panel of sera derived from the Women's Health Initiative cohort (samples obtained 6-18 months prior to the development of breast cancer) and case matched controls. We evaluated serum for the presence of IgG and IgM specific autoantibodies to Pdhx, Otud6b, and Stk39. With both IgG and IgM ELISA analysis, a panel combining the three antigens demonstrates an AUC of 0.633 in patients diagnosed within 5 month and an AUC of 0.688 in patients diagnosed within 5-18 month of the blood draw. In summary, we conclude that tumor antigens identified in neu transgenic mouse model can be used for early detection and diagnosis of breast cancer in women. The combination of antigen specific IgG and IgM antibodies may enhance the sensitivity of the assay for early detection.

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 2683. doi:10.1158/1538-7445.AM2011-2683
Abstract 2684: Immunosensitization of apoptosis-resistant MART-1 negative human melanomas by bortezomib
Authors: Jazirehi Ali R. , Economou James S.
Published: June 2012

Adoptive cell transfer (ACT) of ex-vivo engineered autologous lymphocytes encoding high-affinity MART-1+/ HLA A*0210+-specific T-cell receptor α/β (TCRα/β) (F5 CTL) mediates dramatic, but partial, clinical responses in lymphodepleted metastatic melanoma patients. Various mechanisms have been postulated for the limited clinical responses. Selective outgrowth of antigen (MART-1)-loss variants is one potential mechanism of immune-resistance. Our first hypothesis is that development of MART-1-loss variants is due to MART-1 down-modulation upon encountering F5 CTLs. We also hypothesized aberrant apoptotic signaling may contribute to immune-resistance. To test these hypotheses, we established an in vitro model of resistant (R) lines from sensitive parental (P) lines under F5 CTL-selective pressure. Unlike P lines, F5 CTLs failed to recognize and kill R lines. Moreover, R lines exhibited hyper-activation of NF-κB anti-apoptotic signaling pathway and over-expression of NF-κB-dependent resistant-factors. Specific pharmacological inhibition of NF-κB and silencing of resistant-factors (siRNA) were required, but insufficient, to restore sensitivity of R lines. MART-1 peptide pulsing restored recognition (cytokine release) but not killing of R lines by F5 CTLs. However, bortezomib blocked NF-κB activity, reduced expression of resistant-factors, restored MART-1 expression, and immuno-sensitized R lines. Moreover, MART-1 specific CTLs from metastatic melanoma patients were unable to kill the R lines, whereas bortezomib sensitized the R lines to MART-specific CD8 cells. These novel results suggest that bortezomib interferes with both deregulated signaling pathway and restores recognition of poorly-immunogenic resistant-variants by melanoma-reactive CTLs, thus, imposes a pro-apoptotic/MART-1+ phenotype to R cells and reverses their acquired-resistance. Results also imply that immune-resistance is multi-faceted; development of MHC-/Ag-loss variants and perturbed apoptotic signaling pathways are both important contributing factors. Combining targeted therapy with F5 CTL overrides resistance, which may improve clinical responses.

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 2684. doi:10.1158/1538-7445.AM2011-2684
Abstract 2686: Anti-tumor effects of a folate-immunoglobulin conjugate are enhanced by cytokine treatment in vitro and in vivo
Authors: Jaime-Ramirez Cristina , Kondadasula SriVidya , Jones Natalie B. , Mani Aruna , Roda Julie , Karpa Volodymyr , Lu Yanhui , Li Hong , Zhang Xiaoli , Jarjoura David , Lee Robert J. , Carson William E.
Published: June 2012

Folate conjugation is a means of selectively targeting therapeutics to folate receptor (FR)-expressing cancer cells. A novel folate-bound immunoglobulin (F-IgG) was tested for its ability to target natural killer (NK) cells to folate-receptor expressing cancer cells in the presence or absence of NK-activating cytokines.

FR expression by the KB and HeLa cell lines was confirmed by immunoblot analysis (IB) and flow cytometry. Binding of F-IgG to NK cell Fc receptors led to increased phosphorylation of the epidermal growth factor related kinase (ERK) as measured by IB. Lysis of FR+ KB tumor cells by NK cells was increased 8-fold following treatment with F-IgG as compared to C-IgG (p<0.0001 across E:T ratios from 6.25:1 to 50:1). NK cell lysis of F-IgG- coated KB target cells was significantly enhanced following treatment of NK cells with IL-2, IL-12, IL-15 and IL-21 (all at 10ng/mL). NK cell production of IFN-γ, RANTES and MIP-1α was significantly enhanced by IL-12 in response to F-IgG-coated KB target cells as compared to control-treated cells (p<0.005; IFN-γ-2100 vs. 1000pg/mL, RANTES-800 vs. 150pg/mL, MIP-1α 1800 vs. 500pg/mL, respectively). Studies using the L1210JF murine leukemia model confirmed the anti-tumor activity of F-IgG and the ability of NK-activating cytokines to significantly enhance its effects. NK cell depletion in tumor-bearing mice demonstrated that the anti-tumor effects of IL-12 and F-IgG are dependent on NK cells.

These studies indicate that F-IgG induces an immune response by NK cells against FR-positive cancer cell lines and that cytokine treatment has a synergistic effect on this response both in vitro and in vivo. Thus F-IgG has a potential to be used as a therapeutic antibody for the treatment of FR-positive cancers in combination with immune modulatory cytokines.

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 2686. doi:10.1158/1538-7445.AM2011-2686
Abstract 2700: Effects of anticancer drugs on the viability and the function of peripheral blood mononuclear cells
Authors: Sakai Hiromi , Kokura Satoshi , Ishikawa Takeshi , Okajima Manabu , Matsuyama Tatsuzou , Matsumoto Tsuguhiro , Funaki Jun , Adachi Satoko , Matsuura Yuko , Konishi Hideyuki , Naito Yuji , Yoshikawa Toshikazu
Published: June 2012

[Background/Purpose] Cytotoxic chemotherapy is generally considered immunesuppressive because of its toxicity for dividing cells in the bone marrow. However, recent experimental studies support the notion that certain cytotoxic anticancer agents may actually exhibit immune modulatory activities. Effects of anticancer drugs on adoptive transferred cells remain unclear. Therefore, the purpose of this study was to evaluate the effects of anticancer drugs on the viability and the function of peripheral blood mononuclear cells (PBMCs).

[Method] 5-FU, CPT-11, CDDP and GEM were assessed in the present experiment. PBMCs were treated with each drug for 2hrs, and then cell viability was determined by trypan blue staining and WST-8 assay. For assessment of effects of each drug on proliferation and cytokine producing ability of PBMCs, PBMCs were stimulated with PHA for 48 hrs, following the treatment with each anticancer drug for 2hrs. Proliferation potency of PBMCs was measured using BrdU cell proliferation assay and the producing ability of various cytokines from PBMCs was measured using ELISA. The percentages of IFN-γ-expressing cells and regulatory T cells (Tregs) in GEM-treated PBMCs were measured using flow cytometric assay.

[Results] The anticancer drugs did not affect the cell viability of PBMCs. While 5FU and CPT-11 did not influence proliferation potency, CDDP and GEM inhibited it in concentration-dependent manner. Treatment with GEM enhanced the production of IFN-γ from PBMCs and decreased the number of Tregs induced by PHA. Flow cytometric assay revealed that GEM treatment increased the number of IFN-γ-producing cells in CD4 cells, but GEM did not affect in CD8 cells.

[Conclusions] The anticancer drugs did not affect the cell viability of PBMCs in the present experiment. We have found that GEM may be a practical strategy for the enhancement of IFN-γ production from CD4 cells and reduction of Tregs. Thus, GEM should be evaluated in conjunction with a variety of immunotherapy 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 2700. doi:10.1158/1538-7445.AM2011-2700
Abstract 2713: Association of HBV/inflammation and stemness expression in HBV-related HCC recurrence
Authors: Chang Te-Sheng , Lee Kam-Fai , Lin Hsin-Yi , Tung Shui-Yi , Kuo Liang-Mou , Jen Xin-Wei , Huang Yen-Hua
Published: June 2012

Hepatocellular carcinoma associated with chronic HBV hepatitis (HBV-HCC) tends to have a poorer prognosis. Inflammation is a common and strong participant in the development of HBV-HCC. However, the mechanism of HBV/inflammation-induced hepatocarcinogenesis still remains largely unknown. Here we demonstrate the potential synergistic role of HBV and proinflammatory cytokines in stemness expression which might associate with HBV-HCC recurrence. In this study, human HCC cell lines including HBV- HCC cells (HepG2.2.15, PLC5, Hep3B, and HA22T), and non-HBV HCC cells (HepG2 and Huh 7) were cultivated with inflammation condition medium (inflam-CM). Inflam-CM significantly increased the expression levels of pluripotent transcription factors (Oct-4, Sox2, and Nanog) and the percentage of side population cells in HBV-HCC but less or not in non-HBV HCC. Furthermore, flow-cytometry and epifluorescence analysis showed that inflam-CM markedly increased the GFP+ cell population in Oct-4 promoter-GFP-HepG2.2.15 cells but not in Oct-4 promoter-GFP-HepG2 cells, supporting the synergistic effect of proinflammatory cytokines and HBV in Oct-4 expression. To test the role of stemness expression in HCC prognosis, HA-Oct-4-HepG2 (HepG2 cells over-expressing HA-Oct-4) were used for drug sensitivity and migration/invasion assay. HA-Oct-4 HepG2 cells showed higher drug resistance and migration/invasion ability comparing with HepG2 cells. The responsible proinflammatory cytokines were to be identified using specific neutralizing antibodies and/or signaling inhibitors. The clinical impact was further analyzed using human HCC frozen tissues and tissue blocks which were collected under IRB approval. Real-time quantitative PCR analysis of HCC frozen tissues (N = 102) revealed a significant difference between HBV-HCC and HCV-HCC for the expression of Oct-4 (p=0.0089), Nanog (p=0.0021) and disease-free interval (DFI) (p<0.001). Immunohistochemical staining of HCC tissue blocks (N = 33) further confirmed the strong correlation between stemness expression (Oct-4 and Nanog) and short DFI. In summary, proinflammatory cytokines and HBV may work collaboratively to activate the stemness expression which results in early HBV-HCC recurrence. This finding characterizes a potential target for adjuvant therapy against HBV-HCC.

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 2713. doi:10.1158/1538-7445.AM2011-2713
Abstract 2718: Longer duration of preceding high-risk HPV infection is associated with an increase of chromosomal aberrations in high-grade cervical intraepithelial neoplasia
Authors: Steenbergen Renske D.M , Bierkens Mariska , Wilting Saskia M. , van Wieringen Wessel N. , van de Wiel Mark A. , Ylstra Bauke , Meijer Chris JLM , Snijders Peter J.
Published: June 2012

High-grade cervical intraepithelial neoplastic (CIN2/3) lesions represent a heterogeneous disease both with respect to clinical behaviour and chromosomal aberrations detected. We hypothesized that the extent of chromosomal aberrations reflects the duration of their existence. To test this hypothesis, chromosomal profiles were determined of CIN3 lesions of women with a known 5 year history of high-risk human papillomavirus virus (hrHPV) infection, in which duration of prior hrHPV infection was considered a proxy for duration of CIN3 lesion existence. Eight women had a <5 year preceding hrHPV infection (CIN3<5yrPHI) and 24 had a PHI lasting ≥5 years (CIN3≥5yrPHI). For comparison, 6 CIN3 lesions adjacent to squamous cell carcinomas (CIN3-SCC), their corresponding SCCs, and 6 CIN1 lesions were included. Unsupervised hierarchical clustering analysis of the chromosomal profiles revealed two clusters. One was characterized by relatively quiet genomic profiles including all CIN1, 75% of CIN3<5yrPHI and 37.5% of CIN3≥5yrPHI. Samples in the second cluster, displaying multiple aberrations, included 62.5% CIN3≥5yrPHI and all except one CIN3-SCC and all SCCs. The number of genomic aberrations increased according to lesion grade and also with longer duration of PHI. The increase in aberrations in CIN3≥5yrPHI compared to <5yrPHI was highly significant (p=0.002), suggesting that CIN3≥5yrPHI represent more severe lesions. In conclusion, a longer duration of preceding hrHPV infection is associated with an increased number of chromosomal aberrations. Hence, CIN3 lesions with a longer duration of existence are more prone to have an increased short-term risk of cervical 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 2718. doi:10.1158/1538-7445.AM2011-2718
Abstract 2723: Host response to HMTV
Authors: Melana Stella M. , Fernandez-Cobo Mariana , Marin Tania , Nartey Teiko , Lee Ana , Moran Herberth , Holland James F. , Pogo Beatriz GT
Published: June 2012

Work from our laboratory demonstrated the presence of retroviral sequences homologous to the mouse mammary tumor virus (MMTV) in a large proportion of human breast cancers. These observations have been confirmed by others. A 660 bp sequence of the MMTV env gene, with no significant homology to any viral or human sequence reported in the GenBank, has been found in 40% of the U.S. human breast cancers studied. This sequence is expressed in most of the env positive tumors, and is not detected in the normal breast tissue of patients with env positive carcinomas, indicating its exogenous origin. A complete provirus structure with 95% homology to MMTV has been described and designated as human mammary tumor virus (HMTV). Betaretroviral particles from primary cultures of breast cancer cells (MSSM) have been isolated and characterized. Virion RNA is more than 90% homologous to MMTV RNA and to the HMTV proviral DNA previously identified.

To find out if these particles were infectious, co-culture experiments between MSSM cells and normal human epithelial breast cells, B and T human lymphocytes and human dendritic cells were performed. The results demonstrated that the recipient human cells became infected with HMTV. However, protein expression was only observed in 10-15% of the infected cells by FACS analysis suggesting the presence of an innate resistance to HMTV in human cells. We have then investigated whether human APOBEC and TRIM proteins are implicated in resistance to HMTV expression. The results indicated that APOBEC G and F, TRIM 5, 21 and 25 are all highly expressed in HMTV cells as measured by quantitative RT-PCR. Additional molecular changes seen in infected cells are disruption of the cytoskeleton and evidence for epithelial-mesenchymal transition (EMT).

Since expression of an endogenous retrovirus (HERV-K) env gene has been reported to be upregulated in human breast cancers we have also investigated its expression in HMTV-infected cells, primary MSSM cells and breast tumors by quantitative RT PCR. The results indicated that in all instances where HMTV was present, expression of HERV-K (HML-2) was less than the control counterpart.

In conclusion, HMTV is able to infect a variety of cells bringing about striking molecular changes. Whether these changes play a role in tumorigenesis remains to be shown.

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 2723. doi:10.1158/1538-7445.AM2011-2723
Abstract 2729: Proteomic analysis reveals a network of inflammatory genes in epidermis associated with skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice
Authors: Shen Jianjun , Abel Erika L. , Riggs Penny K. , Angel Joe M. , Repass John , Hensley Sean C. , Schroeder Lisa , Traner Angelina , Chau Alexander , Ward Michael , Semmes O. John , Person Maria D. , DiGiovanni John
Published: June 2012

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with tumor promotion susceptibility by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a proteomic approach of two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. In these experiments, we examined epidermal protein lysates and identified proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following TPA treatment. Among 19 differentially expressed proteins identified using this methodology were two calcium-binding proteins, S100A8 and S100A9. Their differential expression was further examined and validated by one or more of the following methods: i) one-dimensional (1-D) Western blot analysis; ii) 2-D Western blot analysis; iii) immunohistochemical analysis; and iv) quantitative real-time PCR. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially up-regulated in epidermis of DBA/2 vs C57BL/6 mice following topical treatment with two other tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that S100A8/A9 could be linked to several inflammatory networks. Further analyses revealed significantly increased expression of several inflammation-related genes including TNF-α, NFκB and IL-22 in epidermis of TPA-treated DBA/2 mice. Follow-up studies confirmed that these three inflammation related genes were upregulated in epidermis of TPA-treated DBA/2 mice compared to similarly treated C57BL/6 mice. These data suggest that differential expression of inflammation related genes in epidermis contributes to TPA-induced inflammation and skin tumor promotion susceptibility in DBA/2 mice. Taken together, our present data provide further insight into potential molecular mechanisms for the differential susceptibility of DBA/2 and C57BL/6 mice in terms of both inflammation and skin tumor promotion.

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 2729. doi:10.1158/1538-7445.AM2011-2729
Abstract 2735: Osteopontin mediates tumorigenic transformation of a murine promotable cell line through promoting cell survival by an RGD-dependent suppression of caspase 8 activity
Authors: Hsieh Yu-Hua , Chang Pi-Ling
Published: June 2012

Osteopontin (OPN) is a cell adhesive, matricellular protein shown to be an important rate-limiting factor in tumorigenesis in vivo. Further, we have previously shown that addition of exogenous OPN promoted colony formation in an anchorage-independent growth assay of JB6 Cl41.5a cells, a preneoplastic mouse promotable cell line. In addition, down-regulation of the TPA (12-0-tetradecanoylphorbol-13-acetate)-induced OPN expression markedly suppressed TPA-induced colony formation of these cells. Collectively, these findings support the role of osteopontin in mediating neoplastic transformation of JB6 cells in vitro. In the present study, we hypothesize that induced-OPN stimulates the proliferation and/or prevents anoikis of JB6 cells grown in soft-agar and consequently, facilitates colony formation. To determine whether OPN promotes cell proliferation, cell cycle analyses by flow cytometry and direct cell counting of JB6 cells treated with soluble OPN were performed. Data from both of these analyses indicated that OPN-treated cells did not promote cell proliferation compared to non-treated controls. To determine whether OPN prevents anoikis of JB6 cells, annexin V and caspases activities were assesses on cells incubated with or without OPN and suspended on agar. Annexin V analyses showed that OPN significantly decreased anoikis of JB6 cells at 24 h by 41%. In the absence of OPN, suspended JB6 cells underwent anoikis through a sequential activation of caspases 8, 9 and 3 by 1, 3 and 18 h, respectively. Also, the addition of soluble OPN to suspended JB6 cells significantly suppressed caspase 8 activation when compared to non-treated cells. We next determined whether OPN's ability to suppress caspase 8 activity was mediated through its RGD- and/or DRVLFRI-binding motifs as they are known to transduce signals through integrin and CD44 receptors, respectively. Incubation of soluble RGD peptides with suspended JB6 cells suppressed caspase 8 activity. Similar results were obtained when suspended JB6 cells were incubated with OPN only or with RGD peptides and OPN. In contrast, RGE (non-specific peptides) and DRVLFRI peptides alone did not suppress caspase 8 activity of suspended JB6 cells unless OPN was added to these peptides. These findings suggest that OPN suppression of caspase 8 activity of suspended JB6 cells is mediated through its RGD cell-binding motif interacting with integrin receptors. In conclusion, osteopontin promotes tumorigenic transformation of JB6 cells by enhancing the survival of these cells through its RGD-dependent suppression of caspase 8 activity.

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 2735. doi:10.1158/1538-7445.AM2011-2735
Abstract 2737: The MUC1 membrane-bound mucin is an actor in renal tumor progression
Authors: Bouillez Audrey , Gnemmi Viviane , Butruille Caroline , Ringot Bélinda , Hémon Brigitte , van Seuningen Isabelle , Zini Laurent , Leroy Xavier , Aubert Sébastien , Perrais Michael
Published: June 2012

MUC1, an O-glycoprotein membrane-bound mucin, is overexpressed in clear-cell carcinomas (cRCC) with correlation to two prognostic factors, tumor-node metastasis stage and nuclear Fürhman grade. Previously (Aubert et al, Cancer Res 2009), we have shown that MUC1 was significantly overexpressed in metastatic cRCC vs non-metastatic cRCC and MUC1 is a target gene of HIF-1 transcription factor which is a part of the hypoxia pathway, the main renal carcinogenetic pathway. To better understand the roles of MUC1 in cRCC, we used two renal cell lines expressing MUC1 (Caki-2) or not (ACHN cells). Caki-2 cells were stably transfected with shRNA targeting MUC1 and ACHN cells with an expressing vector containing MUC1 cDNA. Flow cytometry assay and confocal microscopy showed a homogenous membrane expression of MUC1 but also in the nucleus. In vitro studies underlined MUC1 role in the invasion, migration and anchorage-independent growth. In contrast, MUC1 had no role in proliferation and clonogenicity. Cellular agreggation test showed that MUC1 expression is associated with a decrease of cell-cell interactions. Phospho-JNK and phospho-p38 levels were higher in MUC1 expressing cells. Transcriptomic analysis results performed on 44K Agilent Microarrays suggest that MUC1 is a candidate in chemoresistance. Subcutaneous xenograft will be also performed. Our results show that MUC1 plays a role in biological properties of renal cancer cells suggesting the important function for this mucin in tumor progression and confirms its potential as a therapeutic target in this type of 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 2737. doi:10.1158/1538-7445.AM2011-2737
Abstract 2744: SNP-SNP interaction between a microRNA and its target gene increases risk of non-melanoma skin cancer
Authors: Nelson Heather H. , Marsit Carmen J. , Kuriger Jacquelyn K. , Karagas Margaret R.
Published: June 2012

Functional genetic variation in microRNA's has the potential to broadly impact disease processes given the large number of gene targets per microRNA. To date, there are only a few examples of functionally verified mirSNPs; among them is rs2910164 contained in the pre-mir-146a. mir-146a is an important modulator of immune responses, and therefore is of interest for non-melanoma skin cancer (NMSC), a highly antigenic cancer. Using a large population-based case control study of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) in New Hampshire, we investigated the impact of this mirSNP on cancer risk, and potential interaction with one of its gene targets (RNASEL, rs486907). While there was no observed risk associated with the C allele of the mir-146a SNP (ORBCC = 0.91, 95% CI 0.74 – 1.10), ORSCC=1.06, 95% CI 0.86 – 1.30), there was a significant interaction between sunburn history and the mirSNP for BCC (pinteraction=0.002). In addition, there was strong interaction between the mir-146a SNP and genetic variation in its target gene RNASEL (BCC, pinteraction=0.01, SCC pinteraction=0.003). Importantly, our study is among the first to demonstrate snp-snp interaction in a microRNA and its target gene. Further, these data imply that RNASEL, an enzyme involved in cellular and viral RNA turnover, is controlled by this miRNA and that this process is important in skin cancer etiology.

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 2744. doi:10.1158/1538-7445.AM2011-2744
Abstract 2770: UGT2B17 copy number variation and the association with serum glucuonides and prostate cancer risk
Authors: Grant Delores J. , Hoyo Cathrine , Oliver Shannon , Gerber Leah , Taylor Loretta , Newman Kathryn , Shuler Katie , Calloway Elizabeth , Livingston Jonathan N. , Richardson Ricardo M. , Schildkraut Joellen , Freedland Stephen J.
Published: June 2012

Prostate cancer development and treatment has been associated with steroid hormones and/or steroid metabolites. While consensus seems to be building that serum testosterone levels are not related to disease risk, the role of genes involved in steroid metabolism have not been extensively studied. The UDP-glucuronosyltransferase (UGT) enzymes are part of a superfamily of genes involved in phase II detoxification of a wide variety of endogeneous and exogenous substances including bile, odorants, steroids, and drugs. The UGT2B17 gene is expressed in the prostate where it catalyzes the conjugation of a glucuronic acid moiety to steroids such as androstane-3α, 17βdiol. Several studies have examined whether a copy number variant (CNV) of UGT2B17 is associated with prostate cancer risk in case control studies, however, results have been inconclusive, and only one of the studies included African Americans. We examined the relationship of UGT2B17 CNV with prostate cancer risk aggressiveness in a hospital-based, case-control study conducted at the Durham Veterans Administration Hospital, and the relationship between serum levels of the glucuronide of androstane-3α, 17βdiol, AAG, in healthy Caucasians and African Americans. Levels of AAG were measured using ELISA. UGT2B17 genotype was determined by gene-specific copy number assays using Taqman Realtime PCR which showed 0, 1, or 2 alleles of the gene. Whereas CNV was not related to race (p=0.85), a higher CNV was significantly associated with higher androstane-3α, 17βdiol levels in healthy white men (p=0.018) but not non-white men (p=0.94). We found no association between CNV and cancer risk among all men (p=0.59) nor when stratified by white (p=0.70) vs. non-white (p=0.72). Among men with cancer, CNV was not related to Gleason score (p=0.32). These findings suggest that CNV may be associated with serum hormones levels, and these associations may differ between white and non-white men. Serum levels may be altered by other genetic or epigenetic factors in non-white populations. The role of CNV as a risk factor for prostate cancer requires further study in larger samples and with genetic and epigenetic variants. Funded by Department of Defense, NIH NCMHD P20 MD000175, S06-GM008049-33

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 2770. doi:10.1158/1538-7445.AM2011-2770
Abstract 2773: Junction plakoglobin gene polymorphisms in prostate cancer and benign prostatic hyperplasia
Authors: Liu Jan , Chang Inik , Singh Kamaldeep , Dahiya Angela V. , Majid Shahana , Saini Sharanjot , Zaman Mohd S. , Chen Yi , Shahryari Varahram , Sandhu Herman , Yamamura Soichiro , Suh Seong O. , Deng Guoren , Tanaka Yuichiro
Published: June 2012

Junction plakoglobin (JUP) is a member of the catenin family that forms complexes with cadherins and these molecules play a role in cell adhesion and motility. Prior studies have demonstrated genetic alterations of the JUP gene to be a risk for diseases. In prostate, expression of JUP has been shown to be down-regulated in the carcinogenesis process and mutations of this gene may play a role in this process. In this study, we hypothesize that polymorphic variants of the JUP gene can be a risk factor for prostate cancer. To test this hypothesis, the genetic distributions of five JUP polymorphisms were analyzed in 131 normal healthy subjects, 132 benign prostatic hyperplasia (BPH), and 177 sporadic prostate cancer samples by sequence-specific PCR and sequencing. The polymorphic sites evaluated were codon 697 (Met to Leu, rs1126821) along with four locations in the promoter region at the following base pairs prior to the ATG start site: -3633 (C to G, rs12450138), -5971 (A to C, 11650969), -9598 (T to C, rs4479305), and -14959 (G to A, rs12937988). Results of these experiments demonstrate that the variants at the -5971 (P<0.02) and -9598 (P<0.07) sites are associated with prostate cancer when compared to normal controls. Odds ratio and 95% confidence interval for cancer were 2.55 and 1.37-4.75, respectively, for -5971 C/C genotype compared to wildtype; and 2.11 and 1.14-3.91, respectively, for -9598 C/C compared to wildtype. Likewise, the variant allele was significantly associated with prostate cancer for both polymorphic sites (P<0.02). Linkage between the -5971, and -9598 sites were observed (D’ = 1). Haplotype analyses show the bases A and T, representing wildtype at the -5971 and -9598 sites, respectively, to be significantly reduced in prostate cancer (P<0.01). However, no differences were observed for the Met697Leu, -3633, and -14959 polymorphic sites for prostate cancer. Also, BPH did not associate with any of the polymorphic sites as compared to healthy controls. This is the first report that evaluates JUP polymorphism in prostate cancer and these results are important in understanding their role in this disease.

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 2773. doi:10.1158/1538-7445.AM2011-2773
Abstract 2789: Discovery of a novel specific MEK and Raf inhibitor, CH5126766 (RO5126766), hit to lead study of a unique scaffold for kinase inhibitor to a clinical compound
Authors: Iikura Hitoshi , Aoki Toshihiro , Hyoudoh Ikumi , Furuichi Noriyuki , Watanabe Fumio , Ozawa Sawako , Sakaidani Masahiro , Matsushita Masayuki , Shimma Nobuo , Harada Naoki , Tomii Yasushi , Aoki Yuko , Takanashi Kenji
Published: June 2012

Background: Among tumor signaling pathways, the most frequently dysregulated in human cancer is Ras-Raf-MEK axis, and therefore allosteric MEK inhibitors and ATP competitive Raf inhibitors are being tested in clinical trials. However, these inhibitors result in marked activation of upstream signal of MEK in tumor cells, especially with mutant K-ras. This feedback activation or activation of Raf would attenuate the anticancer effects of these inhibitors. One of the strategies to prevent this attenuation will be blocking upstream signal concurrent with MEK inhibition. To validate the hypothesis, we executed a hit to lead study of our HTS hit showing MEK/Raf dual inhibition.

Methods and Results: To increase direct MEK inhibitory activity (by binding to MEK) of the HTS hit coumarin compound and indirect Raf inhibitory activity (by enhancement of the binding between B-Raf /C-Raf and MEK1), introduction of a sulfamide group afforded the best result, compared with similar functional groups such as urea, amide and sulfonamide. Fluorine-scan (C-H/C-F substitutions) and nitrogen-scan (CH/N substitutions) worked efficient to increase both MEK and Raf inhibitory activities and optimize physicochemical properties (water solubility, hERG inhibition, CYP inhibitions etc). These three key transformations afforded CH5126766, which has MEK1 (IC50 = 160 nM) and B-Raf (IC50 = 19 nM) inhibitions and excellent PK profiles (mouse BA 93%, CL 1.1 mL/min/kg, rat BA 66%, CL 0.7 mL/min/kg, monkey BA 82%, CL 0.1 mL/min/kg). The compound showed potent antitumor activity in human cancer xenograft model (HCT116) by once daily oral administration (128% of tumor growth inhibition at a 1 mg/kg dose).

Conclusion: SAR study of our novel type molecule (MEK and Raf dual inhibitor) affords identification of CH5126766. It showed strong antitumor efficacy in vivo, good PK in three animal species and good safety profiles. Thus, CH5126766 is now under clinical investigation with solid tumor 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 2789. doi:10.1158/1538-7445.AM2011-2789
Abstract 2790: Discovery of novel Pim-1 inhibitors
Authors: Xiang Yibin , Hirth Bradforth H. , Liu Jinyu , Metz Markus , Asmussen Gary , Bieman Hans-Peter N. , Fitzgerald Maria , Fremgen Trish , Jancsics Katherine C. , Kothe Michael , Papoulis Andrew T. , Stepp David J. , Wei Ronnie , Skerlj Renato
Published: June 2012

Pim-1 is a proto-oncogene which encodes for the serine/threonine kinase of the same name. Recently, Pim-1 has been implicated in multiple human cancers, including prostate cancer, acute myeloid leukemia and other hematopoietic malignancies. Primarily expressed in spleen, thymus, bone marrow, prostate, oral epithelial, hippocampus and fetal liver cells, Pim-1 has also been found to be highly expressed in cell cultures isolated from human tumors.

As part of our continuing efforts to identify potential protein targets and inhibitors in the area of cancer treatment, we selected Pim-1 target and used fragment drug discovery platform to undertake the efforts in identification of novel inhibitors. Over 1800 fragments have been screened using SPR binding assay, followed by PIM1 kinase inhibition assay. Among many active fragments, we identified an interesting small fragment, 5-bromobenzofuran-2-carboxylic acid (1), which has IC50 of 8.1 uM in the Pim-1 kinase inhibition assay. The X-ray structure of 1 binding to Pim-1 revealed that the oxygen of the benzofuran is positioned towards the hinge site while the carboxylic acid interacts directly with the Lys-67. Guided by the X-ray structures, we generated many more potent inhibitors, among which, compound 2 with an aminicyclohexayl moiety at the 7-position is one of the most potent inhibitor with IC50 of 0.001uM in Pim-1 kinase inhibition assay. X-ray structure of 2 binding to Pim-1 indicated that compound 2 gained additional interactions by directly interacting of its nitrogen atom of the aminocycloheaxyl group with Asp127 as well as interacting through water bridges with Glu170 and Asn 171. Compound 2 has also potent activity in inhibiting Pim-2 with IC50 of 0.004 uM while is highly selective against most kinases in the Ambit kinase panel.

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 2790. doi:10.1158/1538-7445.AM2011-2790
Abstract 2793: A hypermethylated phenotype in anaplastic oligodendroglial brain tumors is a better predictor of survival than MGMT methylation in the EORTC 26951 study
Authors: van den Bent Martin , Gravendeel Lonneke , Gorlia Thierry , Kros Johan M. , Wesseling Pieter , Teepen Johannes , Idbaih Ahmed , Sanson Marc , French Pim
Published: June 2012

We have performed methylation profiling (Illumina Human Methylation 27 beadchip) on 69 anaplastic oligodendrogliomas (AOD) or anaplastic mixed oligoastrocytomas (MOA) and one non-diseased normal brain. Fifty-two of these samples were treated as part of the EORTC 26951 study, the primary objective of which was to compare the overall survival (OS) of patients with AOD or MOA treated with radiotherapy (59.4 Gy) vs. ± PCV chemotherapy. Remaining samples were also AOD or MOA and received similar treatment paradigms. Unsupervised clustering using hierarchical ordered partitioning and collapsing hybrid (Hopach) was performed using all or the 10,000, 5,000, 2,000, 1,000, 500, 200 or 100 most variably methylated CpG loci. In all analyses, we identified two main clusters that were highly stable (determined by Fuzzy clustering). In general, these clusters could be separated by predominantly methylated vs. unmethylated CpG loci. Survival (as determined from the date of resection) in the “hypermethylated” subgroup was markedly better than the survival of the unmethylated subgroup (5. 62 vs. 1.24 years, P<0.0001). In fact, methylation profiling was able to better separate long vs. short survivors than histology, MGMT promoter methylation status (as determined by MLPA), 1p/19q LOH (as determined by FISH) or IDH1 mutation status. Methylation phenotype remained an independent prognostic marker in a multivariate analysis that includes diagnosis, age, sex, 1p19q LOH and IDH1 mutation status. We validated our results on methylation profiling data from the cancer genome atlas (TCGA) by assigning TCGA samples to one of the molecular clusters identified in our dataset. 14/80 and 66/80 samples of were assigned to the “hypermethylated” and “unmethylated” cluster respectively. Similar to the initial “test” dataset, survival between the hypermethylated and unmethylated subgroups were significantly different (2.87 vs. 1.02 years, P<0.0001).

In conclusion, methylation profiling has identified two main subgroups of oligodendroglial brain tumors. These methylation subgroups correlate better with survival than histology, MGMT promoter methylation, 1p19q LOH and IDH1 mutation status. We are currently integrating methylation data with expression data.

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 2793. doi:10.1158/1538-7445.AM2011-2793