bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3817: Initial characterization of the prolactin-inducible protein (PIP) null mouse suggests an immunomodulatory role for mPIP
Authors: Yeganeh Behzad , Blanchard Anne , Nistor Andreea , Myal Yvonne
Published: February 2011

Mouse prolactin-inducible protein (mPIP) is the homologue of the human PIP which is a known marker of both malignant and benign human breast cancer. PIP is shown to be highly expressed and secreted by submandibular, lacrimal and salivary glands. Secreted PIP in saliva has been shown to bind to bacteria suggesting that PIP may play a role in innate host defence. Furthermore, human PIP was shown to be a ligand of the CD4 molecule of T lymphocytes, acting as a potential inhibitor of T cell apoptosis suggesting a role in immune host defence.

To address the role of mPIP in innate and adaptive host defence, we generated a PIP knockout mouse (PIP−/−) by targeted gene disruption. Histological as well as physiological and behavioural analyses were used to study the phenotype of these mice. Microarray was also conducted to assess the global effect of mPIP abrogation on gene expression in submandibular glands.

PIP−/− mice develop normally with no obvious phenotypic differences that distinguish them from wild-type siblings. However, some adult mutant mice exhibit increased proliferative activity in lymphoid tissues, evident with enlarged submandibular lymph nodes. Light microscopy of these submandibular lymph nodes revealed activated germinal centres with an increased number of immune cells displaying high mitotic and apoptotic rates. Additional analyses showed enlarged medullary regions in the thymus of some PIP−/− mice populated with mature lymphocytes and lymphocytic aggregations within the prostate lobes. As well, analysis of blood cell profiles revealed significant increased in the number of monocytes (p<0.005, n=6) in PIP−/− mice compared to their corresponding littermates. Microarray analysis of gene expression of the submandibular gland of PIP−/− mice also showed differential expression of multiple genes such as Xiap, Dmtf1, Runx1t1, Ccnb2, Notch4, Zeb2 genes which have been shown to be associated with immune disease, cancer and cell survival.

In summary our data suggests a role for mPIP in modulation of adaptive and innate immune response in mice. Furthermore, our microarray analysis has revealed novel potential genes and pathways involved in mPIP signalling.

(Supported by: Natural Sciences and Engineering Research Council of Canada)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3817.
Abstract 3823: Platinum anti-cancer drugs attenuate T cell-mediated immune suppression through STAT6/PD-L2
Authors: Lesterhuis W. Joost , Punt Cornelis J.A. , Eleveld-Trancikova Dagmar , Jansen Bas J.H. , Schreibelt Gerty , Boer Annemiek De , Adema Gosse J. , Figdor Carl G. , De Vries I. Jolanda M.
Published: February 2011

It has become evident that many immune inhibitory mechanisms in the tumor micro-environment prevent the development of effective T cell responses against the tumor cells in cancer patients. Therapeutic interventions aimed at blocking these pathways have been shown to result in enhanced anti-tumor immunity, but they lack direct cytotoxic effects. Cancer chemotherapeutic drugs have these direct cytotoxic anti-tumor effects and although for some anti-cancer drugs indirect activation of the immune system via induction of an immunogenic type of tumor cell death has been described, their effect on inhibitory pathways in anti-tumor immunity is unknown.

We investigated the effect of a wide array of chemotherapeutics on dendritic cell (DC) function and on tumor cell immunogenicity.

Using allogeneic and antigen-specific in vitro models, we found that when DCs were matured/activated in the presence of platinum-based chemotherapy, they showed strongly enhanced T cell stimulatory capacity. Programmed death receptor-ligand 2 (PD-L2) expression by DC was markedly reduced upon platinum exposure. The enhanced T cell stimulatory capacity by DC upon platinum exposure was abrogated in the presence of PD-L2 blocking antibodies. This was also observed when the regulator of PD-L2 expression, signal transducer and activator of transcription 6 (Stat6), was knocked down using siRNA.

In addition, we found that STAT6 was dephosphorylated in tumor cells by platinum compounds, leading to marked downregulation of PD-L2, resulting in enhanced recognition by tumor-specific T cells.

In line with these in vitro findings, we found enhanced Th1 cytokine secretion and T cell proliferative responses in cancer patients upon treatment with platinum-based chemotherapy, confirming the in vivo occurrence of platinum-induced immune deviation.

Our findings show that treatment with platinum compounds not only results in enhanced T cell stimulation by DC, but at the same time enhances the sensitivity of the tumor for lysis by cytotoxic T cells. This novel double action of platinum compounds may initiate combinations with other immune stimulatory compounds to increase clinical efficacy.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3823.
Abstract 3826: Neutralization of TGFβ enhances the efficacy of chemotherapeutics in a preclinical model of triple negative breast cancer
Authors: Harper Jay W. , Green Tessa , Munroe Kenneth , Manning Charlene , Tao Jingzang , Simon Kailene , Wakefield Lalage , Hsu Frank , McPherson John , Kaplan Johanne , Gregory Rick , Lonning Scott
Published: February 2011

TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Previously we have demonstrated that neutralizing TGFβ with the antibody 1D11 significantly inhibited metastasis in preclinical models of breast cancer and melanoma, with minimal effects on primary tumor growth. Neutralizing the protumorigenic effects that TGFβ has on the immune system and tumor microenvironment could, hypothetically, enhance the direct anti-tumor effects of chemotherapeutic agents. To test this hypothesis, several studies were conducted testing combinatorial therapies with 1D11 and chemotherapeutics against subcutaneous (SQ) 4T1, a murine model of triple negative breast cancer (TNBC), with subsequent metastasis to the lung. Paclitaxel is currently the front-line therapy for TNBC in the clinic. Combining 1D11 with paclitaxel had little effect on enhancing the efficacy of paclitaxel alone against primary tumor growth. However, the combinatorial therapy of 1D11 and paclitaxel inhibited spontaneous metastasis from the primary SQ tumor to the lung to a greater extent than either agent alone. In the clinic TNBC typically becomes resistant to taxane therapy and another treatment that has shown some promise in taxane-refractory TNBC is the combinatorial therapy of capecitabine and ixabepilone. In the 4T1 preclinical model, both capecitabine and ixabepilone inhibited the growth of SQ 4T1 tumors as single agents, and the combination of the two therapeutics resulted in slightly greater efficacy. However, the addition of 1D11 to capecitabine and ixabepilone resulted in even greater efficacy than with the combination alone. Currently, the effects of neutralizing TGFβ with 1D11 during the course of capecitabine/ixabepilone therapy on metastasis in the 4T1 model are being investigated. Additionally, mechanism of action studies are being conducted to determine the mechanisms by which 1D11 is enhancing the efficacy of paclitaxel and the combinatorial therapy. The data from these chemo-combo studies with 1D11 suggests that therapies that target TGFβ may enhance the efficacy of paclitaxel against TNBC metastasis and may potentiate the efficacy of the combinatorial therapy of capecitabine/ixabepilone in taxane-refractory TNBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3826.
Abstract 3829: Chronic stimulation with IL-15/IL-15Rα complexes leads to NK cell dysfunction
Authors: Elpek Kutlu G. , Rubinstein Mark P. , Bellemare-Pelletier Angelique , Goldrath Ananda W. , Turley Shannon J.
Published: February 2011

IL-15/IL-15Rα complexes induce expansion and activation of cytotoxic lymphocytes including NK and CD8 T cells with higher activity compared to IL-15 alone in vivo, and therefore, have therapeutic potential against cancer and chronic infections. Recent studies demonstrated that continuous treatment with IL-15/IL-15Rα complexes improves the survival of tumor bearing mice, however fail to control tumor outgrowth. Given the limited efficacy of long-term cytokine therapy, we sought to investigate the impact of continuous stimulations with IL-15/IL-15Rα complexes on NK and CD8 T cells in tumor-free mice in vivo. Long-term treatment with IL-15/IL-15Rα complexes induced massive expansion of both NK and CD8 T cells. While the functional capacity of CD8 T cells was enhanced after long-term treatments, NK cells exhibited marked defects in activation, IFNγ production, degranulation, and cytotoxicity. In addition, long-term treatment generated a suppressive environment and resulted in marked increase in Treg and CD11c+Gr1+ cell numbers. However, impaired NK cell function was not due to regulatory T cells or various myeloid cell mediated-suppressive factors. Our results have important clinical implications for the design of immunotherapies and vaccines using multiple doses of immunostimulatory agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3829.
Abstract 3837: PI-3 kinase acts upstream of oncogenic Ras in hematopoietic cells through a cytokine-PLCγ-RasGRP pathway
Authors: Diaz-Flores Ernesto , Goldschmidt Hana , Depeille Philippe , Ng Victor , Roose Jeroen , Shokat Kevan , Braun Benjamin , Shannon Kevin
Published: February 2011

Introduction: KRAS is the most common gain-of-function mutation in human cancer and occur in some myeloid malignancies. Despite extensive data implicating aberrant responsiveness to cytokines in the pathogenesis of myeloid leukemias with hyperactive Ras, the underlying mechanisms are largely unknown. We investigated the biochemical basis of hypersensitivity to granulocyte macrophage colony stimulating factor (GM-CSF), IL-3, and stem cell factor (SCF) in primary cells from Mx1-Cre, KrasG12D mice. Methods: We utilized conventional biochemical techniques, phospho-specific flow cytometry, and small molecule inhibitors to interrogate signaling downstream of the activated GM-CSF and c-Kit receptors. Results: We found that endogenous KrasG12D expression deregulates multiple effectors in primary myeloid lineage cells. Although Ras-GTP levels are markedly elevated in bone marrow cells from KrasG12D mice, these cells only fully activate the Raf/MEK/ERK cascade upon cytokine stimulation. We used a “chemical genetic” strategy to explore the biochemical mechanism linking the activated GM-CSF receptor to oncogenic K-RasG12D and ERK. Surprisingly, inhibitors of type I PI3 kinases (PI-90, PI-103) as well as a p110 γ/α isoform inhibitor (AS605240) interfered with GM-CSF and SCF induced Ras/MAPK activation in wild-type and KrasG12D myeloid cells. These effects of PI3 kinase are mediated through activation of PLC-γ and the RasGRP guanine nucleotide exchange factors. Functional studies confirmed that inhibiting PI3 kinase or PLC-γ blocked KrasG12D-mediated myeloid progenitor colony growth. This PI3 kinase/PLC-γ module also functions downstream of the activated c-kit receptor in lineage- cKit+ Sca1+ (LSK) cells in SCF-induced ERK activation. Conclusions: PI3 kinase, PLC-γ, and RasGRPs function upstream of normal and oncogenic K-Ras to efficiently activate the Raf/MEK/ERK cascade in primary hematopoietic cells. These data infer that PI3 kinase is both an important upstream regulator as well as a major downstream effector of Ras-GTP. Significance: Targeting PI3 kinase in hematologic cancers characterized by hyperactive Ras may have unanticipated biochemical and functional consequences in malignant and normal hematopoietic cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3837.
Abstract 3844: Targeting macrophages as a novel therapeutic approach for malignant pleural mesothelioma
Authors: Kolhatkar Nikita S. , Johansson Magnus , Barbone Dario , Yagui-Beltran Adam , Bueno Raphael , Sugarbaker David J. , Jablons David M. , Broaddus V. Courtney , Coussens Lisa M.
Published: February 2011

Mesothelioma is a life-threatening tumor, induced by inhalation of asbestos fibers that is largely resistant to most chemotherapeutic drugs. Using a combination of immunohistochemistry and flow cytometry, we have evaluated both the presence and composition of leukocytes residing in human mesotheliomas, and have found that 1) epithelioid and mixed mesothelioma tumor subtypes contain higher percentages of CD45+ leukocytes than sarcomatous tumors, and 2) macrophages represent the most abundant immune cell subtype in mesothelioms, regardless of type (31 ± 4.6% of total CD45+). Indeed, the percentage of macrophages in mesotheliomas exceeded that found in other thoracic malignancies thus far evaluated (NSCLC cancer, 9%; esophageal, 4%). In view of recent data indicating that macrophages potentiate cancer development in some settings, and that macrophages can be targeted therapeutically to minimize some aspects of tumor progression, we investigated whether macrophages could be targeted to alter the chemosensitivity of human mesotheliomas. To address this question, we adapted a 3-dimensional (3D) spheroid growth model, enabling heterotypic culture of mesotheliomal cells and macrophages. Our data indicate that mesothelioma chemoresistance can be lowered by co-incubation with macrophages. However, the magnitude of the altered response is dependent upon macrophage phenotype. Macrophage phenotype and bioactivity is modulated by TH1 and TH2 cytokine exposure that in turn regulate either an M1 (IFN-γ & LPS) or M2 (IL-4) phenotype. M1-polarized macrophages increased the response of malignant mesothelioma spheroids to pro-apoptotic chemotherapeutic agents, including gemcitabine. Furthermore, our preliminary data indicate that primary human tumor-associated macrophages, isolated from untreated malignant mesotheliomas, exhibit similar pro-apoptotic effects when polarized with M1 cytokines, indicating that cytokine re-polarization of macrophages in mesotheliomas to an M1 phenotype could enhance efficacy of cytotoxic therapy.

This work was supported by grants from the NCI and a DoD Mesothelioma Program grant PR080717 to Broaddus and Coussens

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3844.
Abstract 3845: The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo
Authors: Fleming Jodie M. , Miller Tyler C. , Quinones Mariam , Xiao Zhen , Xu Xia , Meyer Matthew J. , Ginsburg Erika , Veenstra Timmothy D. , Vonderhaar Barbara K.
Published: February 2011

Introduction: Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from pre-menopausal African-American (AA) or Caucasian-American (CA) breast tissue would affect the tumorigenicity of cancer cells in vitro and in vivo. We chose these two populations because of the well-documented predisposition of AA to develop aggressive, highly metastatic breast cancer compared to CA women.

Methods: The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast-ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested in vitro via soft agar and invasion assays, and in vivo via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry.

Results: Mouse mammary glands humanized with pre-menopausal CA-fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA-humanized glands. Examination of 164 extracellular matrix (ECM) molecules and cytokines from CA-derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast-ECM was isolated; invasion and soft agar assays demonstrated that ER-/PR- cells were significantly more aggressive when in contact with AA ECM, as were ER+/PR+ cells with CA ECM. Using zymography, protease activity was comparatively upregulated in CA ECM. In xenograft models, CA ECM significantly increased the tumorigenicity of ER+/PR+ cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed only 1,759 of ∼8,000 identified were in common. In the AA dataset, proteins associated with breast cancer were primarily related to tumorigenesis/neoplasia, while CA-unique proteins were involved with growth/metastasis. Using a novel mass spectrometry method, 17 biologically-active hormones were measured; estradiol, estriol and 2-methoxyestrone were significantly higher in CA breast tissue.

Conclusions: This study details normal pre-menopausal breast tissue composition, delineates potential mechanisms for breast cancer development, and provides data for further investigation into the role of the microenvironment in cancer disparities.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3845.
Abstract 3857: Hedgehog signaling in primary prostate tumors and associated metastases
Authors: Proctor Jennifer , Travaglione Veronica , Deyneko Igor , Read Margaret , Ross Rob , McGovern Karen , Vessella Robert , MacDougall John
Published: February 2011

Malignant activation of the Hedgehog (Hh) pathway is associated with multiple tumor types. The Hh pathway can promote growth of certain cancers via three modes of malignant activation: ligand-dependent signaling either to the tumor cell itself or between the tumor and its microenvironment, and ligand-independent signaling activated by genetic mutations. In certain cancers, tumor-derived Hh ligand signals in a paracrine manner to the stromal cells in the microenvironment that support tumor cell growth. Using a sensitive immunohistochemistry method for detecting Hh ligand, 83 percent of 105 human prostate tumor samples, including lymph and bone metastases from the same patients, were found to express high levels of Sonic Hedgehog (SHh) ligand. LuCaP 23.1 and LuCaP 35v xenografts were derived from lymph node metastasis from patients with prostate cancer. Paracrine Hh signaling is detected in both xenograft models, where the human tumor cells produce Hh ligand and the surrounding stromal cells respond with Hh pathway activation, as measured by Gli1 levels. IPI-926, a novel, small molecule Hh inhibitor that acts by blocking Smoothened, inhibits signaling between the tumor and stromal cells in both castration sensitive and castration resistant tumor models. Inhibition of Hh signaling with IPI-926 was then evaluated as a potential means to reduce growth of prostate cancer tumors and metastases. IPI-926 significantly delays relapse and slows the growth rate of castration resistant primary human prostate tumors post-docetaxel treatment. Additional preclinical studies with IPI-926 in combination with chemotherapy and targeted therapies in castration resistant tumor models are in progress. These pre-clinical results provide a rationale for testing Hh inhibitors in patients with castration resistant prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3857.
Abstract 3873: Squalene enhanced chemotherapy and radiation therapy sensitivity in mice through the inhibition of DNA damage checkpoint activity
Authors: Nishida Hiroshi , Tatewaki Naoto , Nakajima Yuki , Nakayama-Kawakami Kayoko , Kasai-Eguchi Kiyomi , Anzai Kazunori , Konishi Tetsuya , Ikekawa Nobuo
Published: February 2011

ATM (Ataxia Telangiectasia Mutated) protein kinase plays a crucial role in cellular DNA damage responses. The inhibition of ATM leads to an abolishment of one such signal pathways termed as “checkpoints”. It is expected that the discovery of checkpoint inhibitor will be an effective assistance of anti-cancer therapy. Recent study reported that Wip1, a magnesium dependent protein phosphatase, de-phosphorylates molecules involved DNA damage checkpoint response such ATM (Ser1981). On the other hand, Squalene (SQ) has been thought to assist anti-cancer therapies such as chemo and/or radiation therapy though there is few available data which refer to its detailed mechanisms. We previously showed that SQ enhances the γ-irradiation (IR) sensitivity through the induction of Wip1 in A549 adenocarcinoma cells. It was indicated that IR induced DNA damage checkpoint was abrogated by SQ pre-treatment, and led to the shrinkage of tumor in vivo. Here, we report SQ sensitizes chemotherapy and an advanced radiation therapy, heavy ion (HI) exposure, in mice using allo/xeno-graft model. Mouse Colon-26 and human MKN45 cells (5 × 106 cells/ mouse) were transplanted into Balb/c and SCID mice, respectively. The tumor volumes were estimated by measuring the growing diameters of tumor tissue every day. SQ (200μl, oral administration) was challenged after day-8 and later days. Chemotherapy using DOX (2 mg/kg, i.p.) was conducted in same days with SQ. One-time exposure of HI (Carbon, 290MeV) was performed on the day-8 in NIRS (National Institute of Radiological Sciences, Japan). Marked change of body weight was not observed between the treatments. DOX and HI prevented the tumor growth. SQ itself inhibited tumor growth slightly. The administration of SQ significantly improved the efficacy of DOX and HI on tumor growth. Wip1 protein expression in tumor tissue was detected by western blot analysis, and was elevated by the oral administration of SQ. These results were confirmed in xenograft model using MKN45 cells and SCID mice. Taken together these findings, SQ sensitized chemo/radio therapy through the inhibition of DNA damage checkpoint activity by Wip1. Further in-depth study is required to determine the clinical feasibility of SQ as a sensitizing agent for anti-cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3873.
Abstract 3877: An inhibitor-2 complex regulates cell cycle checkpoints in response to DNA damage
Authors: Sun Mianen , Lin Chenghe , Tang Xi , Xu Bo
Published: February 2011

Optimal DNA damage responses such as cell cycle checkpoints are essential for cell survival and prevention of genetic instability. Comprehensive knowledge on the regulation of cell cycle checkpoints will provide molecular insights into tumorigenesis as well as tumor responses to radiation and chemotherapy. The signal transduction network orchestrated by the ATM protein kinase through phosphorylation of downstream targets is critical to activate cell cycle checkpoints in response to ionizing radiation (IR). Among them, Inhibitor 2 (I-2), a regulatory subunit of Protein Phosphatase 1 (PP1), is phosphorylated by ATM on Serine 43 after IR and required for PP1 activation. However, the detailed mechanism of the signaling network involving ATM, PP1 and I-2 has not been elucidated. In the present study, we show that I-2 phosphorylation is required for activation of the S-phase and G2/M checkpoints. We also identified a complex associated with the phosphorylated form of I-2. Several DNA damage responsive proteins such as ATM, Brca1, Cdc7 and Chk2 are presented in the complex. Further, we demonstrate that Brca1 is required for ATM-mediated I-2 phosphorylation and PP1 activation after DNA damage. We find Brca1 is essential for recruiting I-2 to DNA damage sites, and Brca1 interaction with I-2 increases after DNA damage. This DNA damage-induced Brca1/I-2 interaction further lead to I-2/PP1 dissociation, the process required for PP1 activation after DNA damage. Taken together, our results highlighted that a comprehensive pathway mediated by I-2 phosphorylation in response to DNA damage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3877.
Abstract 3878: Perturbations in the AR-cyclin D1 axis controls lethal tumor phenotypes in prostate cancer
Authors: Augello Michael , Burd Craig , Karch Jason , Knudsen Karen
Published: February 2011

Androgen receptor (AR) signaling is critical to prostate cancer development. It has been well established that AR activation promotes lethal tumor phenotypes including proliferation, survival, invasion, and therapeutic resistance. However, mechanisms by which AR promotes these phenotypes are poorly understood. Previous reports assessing cellular proliferation revealed that the AR-cyclin D1a axis plays a significant role in controlling the response to androgen. While activated AR induces cyclin D1a through mTOR mediated mechanisms, accumulated cyclin D1a serves a negative feedback control function, through the ability of the cyclin to bind and suppress further AR signaling. Importantly, this process is frequently abrogated in human prostate cancer through an alternative splicing event that results in production of cyclin D1b. Cyclin D1b is deficient in its ability to regulate AR, and has been shown in other model systems to harbor significant oncogenic activity. Importantly, we have demonstrated that nearly 1/3 of a large cohort of primary prostate cancer tumors exclusively express cyclin D1b. Thus, it is imperative to determine the molecular and cellular consequence of the alternative splicing event in prostate cancer. Endogenous D-cyclins were eliminated using hormone ablation; subsequent viral-mediated reintroduction of cyclins D1a or D1b allowed for unbiased comparison of cyclin function in the presence and absence of androgen. Gene expression profiling confirmed that D1b is unable to manage AR activity. Concordantly, alteration of androgen-sensitive genes was observed, in particular those associated with migration and invasion. In vitro manipulation of the D1 splicing event confirmed these results. Subsequent modeling of cyclin D1b function revealed that cyclin D1b confers a specific anchorage independence and migration phenotype to AR-positive prostate cancer cells. Thus, these data provide the first evidence that perturbation in the AR-cyclin D1 axis controls lethal tumor phenotypes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3878.
Abstract 3896: Non-canonical regulation of the Hedgehog mediator GLI1 by c-MYC in Burkitt lymphoma cells
Authors: Yoon Joon Won , Xu Ke , Iannaccone Philip M. , Walterhouse David O.
Published: February 2011

Aberrant activation of the oncogene GLI1, a key mediator of the Hedgehog (HH) signaling pathway, is observed in a wide variety of human cancers. Although HH plays a major role in GLI1 transcription and activation via Patched (PTCH) and Smoothened (SMO), there is increasing evidence suggesting that other pathways also regulate GLI1 expression. A recent study suggested that upregulation of c-MYC by the EWS/FLI1 fusion protein in Ewing Sarcoma may play a role in upregulating GLI1. To determine the role of c-MYC in the regulation of GLI1 and whether c-MYC regulates GLI1 independently of the HH signaling pathway, we initiated studies in Burkitt lymphoma cells, which constitutively overexpress c-MYC due to a chromosomal translocation. Using RT-PCR, we showed that BL1647, BL1648, BL2392 and BL2625 Burkitt lymphoma cell lines express c-MYC and HH pathway components, including PTCH1, SMO, and GLI1. Using Western blot analysis, we showed that GLI1 protein levels varied among the different cell lines. We demonstrated a sequence-specific interaction between c-MYC protein and E-boxes in the GLI1 promoter with chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). This interaction resulted in transcriptional activation of a luciferase reporter gene in NIH3T3 and HeLa cells. Treating Burkitt lymphoma cells with the c-MYC inhibitor 10058-F4 downregulated GLI1 mRNA and protein and reduced cell viability as measured by MTT assays. In contrast, inhibition of HH-signaling by cyclopamine did not affect GLI1 expression in Burkitt lymphoma cells and only reduced viability of cells when used at high concentrations, possibly representing non-specific cytotoxic effects. Inhibition of GLI1 by GANT61 suggests that GLI1 functions as an anti-apoptotic factor as measured by Caspase3/7 activity in Burkitt lymphoma cells. Collectively, our data provide evidence that c-MYC may function independently of the HH pathway in Burkitt lymphoma to directly regulate GLI1 in a non-canonical manner and support an anti-apoptotic role for GLI1 in Burkitt lymphoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3896.
Abstract 3907: Emergent properties of EWS/FLI regulation via GGAA-microsatellites in Ewing's sarcoma
Authors: Gangwal Kunal , Enriquez Camille A. , Close Devin , Hill Christopher P. , Lessnick Stephen L.
Published: February 2011

ETS proteins are a family of transcription factors that play important roles in the development of cancer. The Ewing's sarcoma EWS/ETS fusion oncoproteins control a number of cancer-relevant phenotypes in that disease. We recently demonstrated that EWS/FLI, the most common EWS/ETS fusion in Ewing's sarcoma, regulates a portion of its target genes, including the critical target NR0B1, via GGAA-containing microsatellites in their promoters. Given the unusual nature of microsatellites as EWS/FLI response elements, we sought to elucidate the mechanism of EWS/FLI activity at these sites. In addition to EWS/FLI, we found that other Ewing's sarcoma associated EWS/ETS fusions are capable of mediating gene regulation via the NR0B1 microsatellite. Furthermore, regulation via the GGAA microsatellite was specific to Ewing's sarcoma fusion proteins as two other ETS family members, ETS1 and ELF1, could not cause transcriptional activation, highlighting a neomorphic function of the Ewing's sarcoma fusion proteins. The stoichiometry of this interaction is 2 protein molecules for each DNA molecule, suggesting that EWS/FLI binds these elements as a homodimer. The isolated FLI ETS domain bound microsatellite sequences in a nearly identical fashion to full-length EWS/FLI, thus indicating that residues required for homodimeric binding are localized to the ETS domain. These data suggest a new paradigm for ETS family member binding to DNA at cancer-relevant genetic loci, and highlight emergent properties of EWS/FLI that are required for the development of Ewing's sarcoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3907.
Abstract 3910: DLX4 homeprotein enhances the invasiveness of Hs578T ER-negative breast cancer cells
Authors: Fu Yebo , Lian Yi , Kim Kyung Soon , Lee Norman , Fu Sidney W.
Published: February 2011

Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. It was reported that BP1, a homeobox isoform of DLX4, is overexpressed in 80% of breast cancer patients and in 100% of estrogen receptor negative (ER-) tumors. The prevalence of BP1 positive cells and the intensity of BP1 immunoreactivity increased with the extent of ductal proliferation and tumorigenesis. These findings imply that BP1 may play an important role in ER- breast cancer. We sought to determine the effects and mechanisms of BP1 on cell proliferation and metastasis using ER- Hs578T cells as a model. Cells were transfected with either pcDNA3.2 plasmid containing BP1 gene, or pcDNA3.2 vector, then selected and cloned. Overexpression of BP1 increased cell proliferation rate by 2-5 fold (p<0.005), and enhanced the in vitro invasive activity by 25-65 fold (p<0.001). Microarray experiments were performed to identify differentially expressed genes when BP1 is overexpressed. The gene expression profile of the transfected cell lines were compared, resulting in 71 differentially expressed genes with a fold-change of >=2.0. Of those genes, 49 were up-regulated and 22 were down-regulated. Further analysis has identified several important molecular pathways using the differentially expressed gene list, including CREB signaling, BCL2L1, AKT signaling, apoptosis, androgen receptor pathways, etc. CREB1, as an oncogenic transcription factor, induces transcription of genes in response to hormonal stimulation of the cAMP pathway. CREB1 can be activated through phosphorylation by a number of kinases, including AKT, protein kinase A, and calcium/calmodulin-dependent kinases and regulates genes whose deregulated expression promotes oncogenesis, including cyclins, BCL2 family members, and EGR1. It was reported that BP1 can directly bind to and activate the expression of BCL1, which is an antiapoptotic gene and considered to be an oncogene. BCL2L1 is a BCL2 protein family member that acts as anti- or pro-apoptotic regulators involving in a wide variety of cellular activities. These data demonstrated that overexpression of BP1 significantly enhanced cell proliferation and metastatic potential in ER- Hs578T cells. Further analysis with more ER- cell lines and patient samples is warranted to establish BP1 as a therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3910.
Abstract 3918: PEA3 is a novel transcriptional activator of Notch-1 and Notch-4 in breast cancer: Opportunity for a new combination strategy
Authors: Clementz Anthony G. , Rizzo Paola , Miele Lucio , Osipo Clodia
Published: February 2011

Women diagnosed with triple-negative breast cancer have the worst overall prognosis and frequently present with metastatic tumors. To date, there are no targeted therapies available to combat this aggressive form of breast cancer due to the lack of expression of well-known targets such as ERα, PR, or HER2/neu. Therefore, there is an immediate need to identify novel targets that are responsible for the proliferation, survival, and invasive phenotype. Notch-1 and Notch-4, both potent breast oncogenes, are overexpressed in triple-negative breast cancers-associated with the poorest overall survival. PEA3 (polyomavirus enhancer activator 3), a member of the Ets family of transcription factors, is overexpressed in triple-negative breast cancer and also correlates with aggressive behavior and poor overall survival. Here, we provide new evidence for transcriptional regulation of Notch in triple negative breast cancer and other subtypes of breast cancer. With focus on triple-negative breast cancer cells (MDA-MB-231), our results show that PEA3 is a transcriptional activator of both Notch-1 and Notch-4 as determined by using a PEA3 siRNA and measuring both transcripts and proteins. Chromatin immunoprecipitation confirmed enrichment of PEA3 within the promoter regions of both Notch-1 and Notch-4. Notch-1 recruitment appears to be AP-1 independent, whereas PEA3 recruitment on the Notch-4 promoter is dependent upon c-JUN. Furthermore, results showed that either c-Jun or Fra-1 were required for Notch-4 transcription while c-FOS was a repressor. Importantly, the combined inhibition of Notch signaling (via a gamma-secretase inhibitor, GSI) and PEA3 (via siRNA) arrested the cells in G1, decreased cell viability, reduced tumorgenicity in vitro, and increased apoptosis. Taken together, our results indicate that PEA3 is a novel transcriptional activator of both Notch-1 and Notch-4. PEA3-mediated Notch-1 transcription is AP-1 independent while Notch-4 transcription requires both PEA3 and AP-1 most probably composed of the c-Jun: Fra-1 complex. Moreover, both PEA3 and Notch signaling are essential for proliferation and survival of MDA-MB-231 breast cancer cells. Thus, dual targeting of both PEA3 and Notch pathways might provide a new therapeutic strategy for triple-negative breast cancer as well as additional therapeutic targeting in other breast cancer subtypes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3918.
Abstract 3923: ATM and KAP1 are required for nonhomologous end-joining in transcriptionally active chromatin
Authors: Hawkins Amy J. , White David E. , Golding Sarah E. , Akopiants Konstantin , Rauscher Frank J. , Povirk Lawrence F. , Valerie Kristoffer
Published: February 2011

The effect of ATM signaling on nonhomologous end joining (NHEJ) was investigated using a novel, chromosomally-integrated, viral vector that allows for inducing tandem I-SceI-mediated DNA double strand breaks (DSBs). The DSBs can then be analyzed for NHEJ repair events by fluorescence- and PCR-based methods. Using highly specific kinase inhibitors and this repair system, we show that inhibiting ATM reduces NHEJ by 80% in human U87 glioma cells. PCR products that span the repaired DSBs were analyzed by cleavage with a restriction enzyme that exclusively cuts the DNA repaired by high-fidelity repair. This analysis showed that the extent of high-fidelity repair was reduced by 40% when the ATM kinase was inhibited. KAP1 is a DNA damage-induced phosphorylation target of ATM that is linked to the modulation of chromatin structure. The ATM kinase inhibitor used in our studies blocks radiation-induced phosphorylation of KAP1 on serine 823/4. Knocking down KAP1 via shRNA had no effect on homologous recombination repair, but reduced NHEJ by 80%. Simultaneous treatment of cells with the ATM inhibitor and trichostatin A, a histone deacetylase inhibitor that promotes chromatin decondensation, abrogated the effect of the ATM inhibitor. These data are consistent with the hypothesis that ATM regulates DNA accessibility in chromatin. Together, these results suggest that ATM is critical for NHEJ of I-SceI DSBs and for high-fidelity repair of breaks within transcriptionally-active chromatin. The requirement for ATM in this system is likely a consequence of its signaling to chromatin modulating proteins that dynamically act on chromatin architecture surrounding DSBs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3923.
Abstract 3927: Distinct roles of hMLH1-hMRE11 in homologous recombination and heteroduplex DNA repair at a single chromosomal locus
Authors: Her Chengtao , Xu Keqian , Tompkins Joshua D. , Wu Xiling
Published: February 2011

Increasing evidence indicates that the mismatch repair protein hMLH1 and DNA double-strand break (DSB) repair protein hMRE11 play multifunctional roles in various processes of DNA repair and damage response in human cells. Our previous studies have revealed a physical interaction between these two proteins and have suggested a role for hMRE11 in heteroduplex DNA repair and hMLH1-dependent DNA damage-induced G2 arrest. Here, we analyzed the effects of hMLH1-hMRE11 on homologous recombinational repair of an induced DSB and consequential heteroduplex repair at a single chromosomal locus by a newly developed reporter system. The results of these studies indicate that hMLH1 and hMRE11 display a synergistic anti-recombination effect and the hMLH1-mediated anti-recombination is at least partially dependent on its interaction with hMRE11, whereas the repair of heteroduplex DNA requires functional hMLH1 and hMRE11. Consistent with these results, chromatin immunoprecipitation (ChIP) analysis of the reporter locus demonstrates that DSB triggers the loading of hMLH1 and hMRE11 proteins to both the proximal region and the site containing heteroduplex DNA. However, in contrast to the proximal regions, loading of hMLH1-hMRE11 at the site of heteroduplex DNA also requires hMSH2, suggesting the involvement of DNA mismatch repair. In summary, our studies have provided evidence to suggest that, in addition to DNA damage response, hMLH1-hMRE11 are involved in at least two other DNA damage repair processes: anti-recombination and heteroduplex DNA repair. Our results implicate that mutations impairing the hMLH1-hMRE11 interaction might promote spontaneous chromosomal recombination between ectopic repetitive sequences and thereby increasing gene conversion and genomic instability.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3927.
Abstract 3930: Radiation-induced dicentrics: Tracking by parallel sequencing
Authors: Vaughan Andrew T.M , Shih Shyh-Jen , Singh Sheetal , Do To Uyen
Published: February 2011

Radiation and cytotoxic drugs exert much of their lethal effects by creating aberrations, such as dicentrics, that are lethal at a subsequent mitosis. Their accurate enumeration in experimental and clinical settings would provide valuable information on the efficacy of radiation and radiation/chemotherapy combinations. In this study a method was sought that could rapidly and efficiently quantitate this process using next generation sequencing. The use of modern molecular technology for aberration analysis has been restricted by the random nature of the breaks induced in individual chromosome that inhibit their detection. To overcome this problem, a common fragile site at 11q23 that is readily cleaved in response to a broad range of cytotoxic agents was used. Using irradiated human TK6 lymphoblastoid cells and blood samples taken from patients undergoing chemotherapy, partner chromosomes that are ligated to this location were detected using inverse PCR to capture the breakpoint. In order to enhance the efficiency of the process, amplified inverse PCR material was processed for Solexa type parallel sequencing. Here individual amplicons, representing the total content of a treated sample, are attached to a flow cell and their sequence recorded by sequential additions of fluorescent bases. Each aberration was then reconstructed with reference to a standard human genome database. In one specific sample of treated TK6 cells, two translocations involving Chr. 2 and 18 and two dicentrics involving Chr. 3 and 13 were found linked to the Chr 11q23 fragmentation site. In addition, aberrations were found in patients’ blood 6 months after initiation of therapy. Thus translocations that are rarely lethal could be readily discriminated from dicentric type rearrangements that are predominately lethal. The advantage of this analytical process is that all normal sequence is also recorded allowing the fractional content of all aberrations to be recorded in response to dose or time. In order to mitigate concerns over the arbitrary nature of aberrations induced at a common fragile site, a zinc finger nuclease was constructed targeting a region adjacent to the fragile site at 11q23. With this zinc finger system repetitive DNA breaks at the predicted site generated micro rearrangements (insertions/deletions) that were produced at far higher rates than translocations or dicentric type rearrangements measured by parallel processing. In summary, a novel analytical system for detecting a lethal class (dicentrics) of chromosome rearrangements has been described that is suitable for therapy monitoring both in-vitro and in-vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3930.