bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 602: Silencing of the androgen receptor (AR) with a novel mRNA antisense oligonucleotide causes antitumor effects in xenograft models of prostate cancer
Authors: Zhang Yixian , Castaneda Stephen , Kim Steven , Kraft Patricia , Bandaru Raj , Filpula David , Greenberger Lee M. , Horak Ivan D.
Published: February 2011

Background: The AR is a ligand-activated transcriptional factor that plays an important role in prostate cancer. While androgen-deprivation therapies typically inhibit tumor growth, tumor recurrence due to the castration resistant form of the disease frequently occurs and leads to mortality. Nevertheless, castration-resistant tumors are still dependent on a functional (AR). Therefore, inhibition of AR expression, rather than systemic androgen deprivation, may provide a novel strategy for treatment of advanced prostate cancer. We have developed a novel locked nucleic acid (LNA)-based antisense oligonucleotide (ON), EZN-4176, which down-modulated AR and inhibited tumor growth. Methods: The target mRNA down-modulation, growth inhibition, luciferase activity, protein inhibition, and reduction of prostate specific antigen (PSA; an AR downstream target and biomarker of prostate cancer) were evaluated by qRT-PCR, MTS, SteadyGlo, western blot analysis, and ELISA assay, respectively, in LNCaP cancer cell lines. A mismatched LNA antisense oligo (EZN-4176MM) and an AR negative cell line (PC-3 prostate cancer) served as controls. Additionally, the effect of EZN-4176 on DHT-induced AR transcriptional activity was evaluated in LNCaP cells containing an AR-responsive luciferase reporter. In vivo, therapeutic efficacy of EZN-4176 was evaluated in the AR-positive CWR22 (androgen dependent). Results: In vitro, in the absence of any transfection reagent, EZN-4176 but not the mismatched control (EZN-4176MM) inhibited the growth of DHT-induced LNCaP cells. The growth reduction correlated with AR down-modulation and its down-stream target gene expression and AR-responsive luciferase activity. In vivo, EZN-4176, prepared in the absence of a delivery vehicle, demonstrated dose-dependent inhibition of mRNA of AR, PSA, and TMPRSS2 as well as protein level of AR in androgen-sensitive CWR22 tumor xenografts, resulting in significant tumor growth inhibition (TGI) (∼55%) (similar to high-dose Casodex® [bicalutamide] treatment). Cy5.5-labeled EZN-4176 was shown to localize in subcutaneous CWR22 or LNCaP tumor xenografts using xenogen imaging system. In castration-resistant tumor cells, EZN-4176 potently and specifically inhibited the growth of C4-2b cells as well as down-regulated AR-luciferase reporter systems. More importantly, AR and PSA mRNA were inhibited in both castration-resistant C4-2b and 22RV1 tumor xenografts. Conclusions: Our data suggest that AR mRNA down modulation by EZN-4176 inhibits prostate tumor growth and provides a novel strategy to treat advanced 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 602.
Abstract 603: Adenoviral mediated expression of Omomyc induced growth inhibition and apoptosis in lung cancer expressing Myc
Authors: Fukazawa Takuya , Matsuoka Junji , Yamatsuji Tomoki , Maeda Yutaka , Durbin Mary , Naomoto Yoshio
Published: February 2011

The Myc basic helix-loop-helix zipper domain determines dimerization with the constitutively expressed bHLH-Zip protein, Max, and also binding to the DNA E-box. Both of these functions play a critical role in the Myc regulation of growth, proliferation, tumorigenesis, and apoptosis. It has been reported that Myc recognition specificity is encoded in a short region within the leucine zipper. Mutation of four amino acids (E57T, E64I, R70Q, and R71N) generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. As a consequence, Omomyc suppresses activation of artificial E-box promoter elements by Myc/Max and strongly potentiates Myc-induced apoptosis in a manner dependent on Myc expression levels. Furthermore, Omomyc prevents initiation of K-RasG12D-induced lung tumors (Soucek L et al. Oncogene. 17: 2463-72. 1998, Nature. 455:679-83. 2008). In this study we have constructed an adenoviral vector (Ad-Omomyc) for use as a novel therapy for Myc or K-Ras mutated cancer. Ad-Omomyc expresses Omomyc and is driven by the CMV promoter. MTS assay and flowcytometric analysis demonstrated that Ad-Omomyc significantly suppressed cell viability and induced apoptosis in A549 pulmonary adenocarcinoma cells, which express Myc and harbor K-Ras mutations. Moreover, the combination treatment of Ad-Myc and Ad-Omomyc increased apoptosis in H441 pulmonary adenocarcinoma cells harboring a K-Ras mutation as compared to administering Ad-Omomyc alone. Ad-Omomyc induced little apoptosis in normal human lung fibroblasts (NHLF). Here, we describe a novel strategy to target Myc or K-Ras for the treatment of lung cancer using a Myc derivative: Omomyc.

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 603.
Abstract 609: Paradoxical actions of a c-Src inhibitor on estradiol-induced apoptosis in long-term estrogen deprivation breast cancer cells
Authors: Fan Ping , Kim Helen R. , Jordan V. Craig
Published: February 2011

Resistance to aromatase inhibitors used for the treatment of estrogen receptor (ER) positive breast cancer is an increasing problem clinically. We have demonstrated in the laboratory that long term estrogen-deprived MCF-7 breast cancer cells referred to as MCF-7:5C (Lewis et al, JNCI 2005, 97:1746-1759), respond to physiological estrogen by triggering apoptosis. This scientific observation is now in clinical trial, but only 30% of patients receive clinical benefit (Ellis et al, JAMA 2009, 302:774-780). A goal of translational research is to increase the therapeutic responsiveness of antihormone resistant breast cancer. c-Src inhibitors are currently of interest, as they inhibit survival pathways in breast cancer. We have evaluated the potential of the c-Src inhibitor, PP2, alone (5 µM), and compared results of cell survival with estradiol (1 nM E2) alone, or in combination with PP2. The MCF-7:5C cells have increased levels of phosphorylation c-Src, compared to wild type MCF-7 cells. Long term treatment (at least 2 months) with PP2 initially decreased growth rates of MCF-7:5C cells, but then growth recovered. Long term treatment of MCF-7:5C cells with E2 initially causes massive apoptosis, but a population of surviving cells subsequently regrew, but these ER-positive cells were refractory to estrogen action. In contrast, a combination of PP2 + E2 actually retarded apoptosis and the resulting cell line (MCF-7:PF) is unique, as they grew vigorously in culture with physiological levels of estradiol, but remarkably are also stimulated in vitro by a panel of selective estrogen receptor modulators (SERMs). This is the first description of a Phase I (Jordan, Cancer Cell 2004, 5:207-213) resistant breast cancer cell line in vitro. These data illustrate that caution must be exercised when considering the evaluation of c-Src inhibitors in clinical trial following the development of acquired resistance to aromatase inhibitors. A combination with therapeutic estrogen treatment or indeed endogenous estrogen may impair therapeutic effectiveness of c-Src in breast 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 609.
Abstract 612: FOXO1A is a target for HER2 overexpressing breast tumors
Authors: Wu Yanyuan , Shang Xiying , Sarkissyan Marianna , Slamon Dennis , Vadgama Jaydutt V.
Published: February 2011

Trastuzumab treatment has improved the overall survival of HER2 overexpressing breast cancer patients. However, many of these patients will eventually become resistant to treatment. It is unclear what mechanisms may contribute to resistance to Trastuzumab. In this study we have tested the hypothesis that targeting of the FKHR transcription factor FOXO1A in HER2 overexpressing breast tumor cells, can overcome the Trastuzumab resistance in vitro. We have demonstrated that overexpression of HER2 leads to activation of PI3K/Akt pathway and subsequent inactivation of FOXO1A in HER2 overexpressing breast cancer cells, SKBR3. In wildtype SKBR3 cells, Trastuzumab downregulates active Akt (pAkt) and increases FOXO1A expression that leads to increase in p27kip1, and decrease in cyclin D1, and finally inhibits SKBR3 cell proliferation. In contrast, the effect of Trastuzumab was eliminated by the reduction of FOXO1A in HER2 overexpressing cells with constitutively active Akt1 (Clone SKBR3/AA28). The down-regulation of FOXO1A in SKBR3/AA28 resulted in nuclear export of p27kip1. Blocking the constitutively active Akt by a specific Akt/protein kinase B signaling inhibitor-2 (API-2) significantly increased FOXO1A expression and rendered the SKBR3/AA28 cells more responsive to Trastuzumab induced growth inhibition. Re-activation of FOXO1A by stable transfection or transient transfection also restored the growth inhibitory effects of Trastuzumab in SKBR3/AA28 cells. In summary, Trastuzumab can inhibit proliferation of HER2 overexpressing breast cancer cells by re-activating FOXO1A through inhibition the PI3K/Akt pathway. FOXO1A may therefore serve as a target for HER2 overexpressing breast tumors, and its activation or increased expression may overcome resistance to Trastuzumab treatment.

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 612.
Abstract 613: Increased PI3K/AKT activity, which confers resistance to trastuzumab, can be overcome by lapatinib
Authors: O'Brien Neil A. , Wang YuHua , Chow Lucy , Wilcox Karen , Browne Brigid , Crown John , Arboleda Jane , Ginther Charles , Duffy Michael J. , O'Donovan Norma , Slamon Dennis J.
Published: February 2011

Trastuzumab and lapatinib provide clinical benefit to women with HER2 positive breast cancer. However, not all patients whose tumors contain the HER2 alteration respond. There are no clinically validated biomarkers of resistance to therapy. We evaluated responses to trastuzumab and lapatinib in a panel of 17 HER2-amplified cell lines in both two and three dimensional culture and investigated the role of receptor tyrosine kinase (RTK) signaling and PI3K/AKT pathway activation in resistance. We also conditioned four cell lines to acquire trastuzumab resistance, and two additional cell lines to acquire lapatinib resistance. The SUM-225, HCC-1419, HCC-1954, UACC-893, HCC-1569, UACC-732, JIMT-1 and MDA-453 cell lines are innately resistant to trastuzumab, whereas the MDA-361, MDA-453, HCC-1569, UACC-732, JIMT-1, HCC-202 and UACC-893 cells are innately lapatinib resistant. Lapatinib is active in both de novo (SUM-225, HCC-1419 and HCC-1954) and acquired trastuzumab resistant cell lines. Increased phosphorylation of HER2, EGFR, HER3 and IGF-1R correlated with response to lapatinib but not trastuzumab. p95HER2 levels correlated with response to lapatinib, however p95HER2 levels also correlated with levels of full length HER2, indicating that p95HER2 may not be an independent marker of lapatinib response. A decrease in AKT phosphorylation in response to trastuzumab or lapatinib treatment correlated significantly with response to either agent, indicating that loss of pAKT is a biomarker of response to both HER2-targeted therapeutics. Accordingly, trastuzumab treatment of trastuzumab resistant cells had little effect on AKT phosphorylation, whereas treatment of these cells by lapatinib significantly reduced pAKT levels. Thus, lapatinib retains its activity in trastuzumab resistance via continued dephosphorylation of AKT. We also observed that loss of PTEN or the presence of activating mutations in PI3K marked resistance to trastuzumab yet lapatinib response was independent of these factors. Thus, increased activation of the PI3K/AKT pathway confers resistance to trastuzumab which can be overcome by lapatinib. In conclusion, pharmacological targeting of the PI3K/AKT pathway may provide benefit to HER2 positive breast cancer patients who are resistant to trastuzumab 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 613.
Abstract 619: Dual targeted therapies to overcome tyrosin kinase inhibitors resistant in epidermal growth factor receptor (EGFR) expression of lung cancer
Authors: Huang Wen-Chien
Published: February 2011

Background: Non-small cell lung cancer (NSCLS) comprises about 80% of all lung cancers. The regular treatments for NSCLC are surgery, chemotherapy, radiotherapy and EGFR-targeted therapy. EGFR is frequently mutated and amplified in lung adenocarcinom as sensitive to EGFR inhibitors. The EGFR-targeted therapy, Gefitinib and Erlotinib, significantly improves disease free survival. However, it was not possible to predict super-responder of gefitinib degree of EGFR expression as a single agent. There is an imposing need for new rationally designed regimens to overcome Gefitinib resistance. The other tyrosine kinase inhibitor, Lapatinib, is a small-molecule inhibitor targeting both the HER2 and the epidermal growth factor receptor (EGFR) in solid tumors. We hypothesized that this aberrant activation of EGFR may contribute to Gefitinib resistance and dual blocking EGFR activation with EGFR and ERBB2 inhibitor will re-sensitize HER family in lung cancers.

Experimental Design: In the in vitro study, cancer cells with EGFR wild type or mutation type were treated with Tyrosine kinase inhibitor, Gefitinib, Lapatinib, or combined treatments. The responses of cells to Gefitinib or/and Lapatinib treatments were evaluated by MTS proliferation, apoptosis assay, soft agar colony formation assay and western blot. In the in vivo study, pulmonary xenograft tumors were established in ICR/SCID mouse and treated with vehicle control, Gefitinib, Lapatinib, or Gefitinib plus Lapatinib for 4 weeks. Tumor volume were monitored and further studied by immunohistochemistry on collected tumors.

Results: We selected multiple pairs of EGFR wild type and mutation type. The EGFR mutation cells have more effect under single tyrosine kinase inhibitor (Gefitinib) treatment compared with EGFR wild type cells. Dual inhibition of EGFR activation sensitized EGFR expression pulmonary cancer cells to Gefitinib treatment. Compared with single treatment, combined treatment led to a significantly more effective inhibition of cell proliferation and induced apoptosis. In a mouse pulmonary xenograft model, combination therapy with Gefitinib and Lapatinib dramatically inhibited EGFR wild type tumor growth.

Conclusions: Since the EGFR family inhibitor, Lapatinib is currently under Phase III clinical trial, combining Gefitinib with this Lapatinib, dual EGFR inhibitors is a clinically applicable strategy and a promising combinatory targeted anti-cancer therapy to overcome EGFR wild type mediated Gefitinib resistance.

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 619.
Abstract 62: Alteration of metabolic pathways of glucose consumption by CENU treatment in B16 melanoma tumors: A NMR spectroscopy-based [1,2-13C]glucose fluxomics analysis
Authors: Morvan Daniel , Fages Anne , Schwartz Laurent , Steyaert Jean Marc , Stepien Georges , Demidem Aicha
Published: February 2011

We have previously shown using proton NMR spectroscopy-based metabolomics that mice bearing B16 melanoma tumors treated by chloroethylnitrosourea (CENU) had a strong intratumoral decrease in glycolysis involving the accumulation of intracellular glucose and glucose-6-phosphate, together the accumulation of glutamate derivatives, despite no significant lactate content change (1).

To get insights into the metabolic pathway followed by glucose carbons in CENU-treated tumor, we used double labelled glucose ([1,2-13C]glucose) and investigated the obtained isotopomers of lactate, the final product of glycolysis. Mice bearing B16 melanoma tumors were treated by CENU or saline solution (control) at days 11, 14 and 17 at dose 15µg/g weight. At day 23, mice were injected intraperitoneally with [1,2-13C]glucose at 25 mg in 150µl saline solution. Animals were sacrificed 30 minutes after glucose injection. Tumors were removed and kept at −80°C until use. The analysis of lactate isotopomers, a fluxomics approach, was done at 500 MHz using high resolution magic angle spinning (HRMAS) 1H-NMR spectroscopy by exploiting 1H-13C coupling signals. The two lactate signals, the methyl (C3) signal centered at 1.33 ppm and the methine (C2) signal centered at 4.12 ppm were differentially analyzed, and quantified from spectra witout and with broadband 13C-decoupling. In addition, the activity of pyruvate-kinase was measured using enzyme assay.

In comparison with control tumors, CENU-treated tumors exhibited an overall decrease of labelled lactate both at C3 and C2 position, together with a significant increase in the relative proportion of C2 to C3 labelling (0.65±0.10 vs 0.91±0.13, P<0.01, n=3 vs n=3). Because lactate only labelled on C3 was a product of glucose metabolism through the pentose phosphate pathway, it was concluded that most of glucose in CENU-treated tumors was directly metabolized to lactate, thus dedicated to ATP production rather than macromolecular biosynthesis. Consistently, the activity of pyruvate kinase, the main source of glycolysis-derived ATP was increased under CENU treatment (+48%, P<0.01).

Altogether these fluxomics data provide evidence of glycolysis adaptation that may be correlated to reduced proliferation, decreased aggressiveness, and redifferentiation, as previously shown in this model (2). In addition, this study suggests that metabolic targeting of glycolysis-related ATP production may improve CENU efficacy.

1- Morvan D, Demidem A. Cancer Res, 67; 2150-59, 2007

2- Demidem A et al. Cancer Res, 61; 2294-300, 2001

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 62.
Abstract 629: Hepatocyte growth factor induces resistance to an irreversible epidermal growth factor receptor inhibitor in EGFR-T790M mutant lung cancer
Authors: Yamada Tadaaki , Matsumoto Kunio , Donev Ivan Shterev , Sekido Yoshitaka , Nishioka Yasuhiko , Sone Saburo , Yano Seiji
Published: February 2011

Purpose:

Epidermal growth factor receptor (EGFR)-activating mutations, such as deletion in exon 19 and L858R point mutation in exon 21,were found in non-small cell lung cancer and associated with favorable response to the reversible EGFR tyrosine kinase inhibitors (EGFR-TKI), gefitinib and erlotinib. However, almost all NSCLC patients with EGFR mutations who show response to reversible EGFR-TKI ultimately develop resistance to these agents.

Recently, several mechanisms were reported to induce acquired resistance to reversible EGFR-TKI for non-small cell lung cancer with EGFR-activating mutations. Of these, the secondary T790M mutation in EGFR is the most frequent cause of acquired resistance to the reversible EGFR-TKI.

Irreversible EGFR-TKIs are expected to overcome the reversible EGFR-TKI resistance of lung cancer harboring T790M mutation in EGFR. However, it is clear that resistance may also develop to this class of inhibitors. We showed previously that hepatocyte growth factor (HGF) induced gefitinib resistance of lung cancer harboring EGFR-activating mutations.

Here, we investigated whether HGF induced resistance to the irreversible EGFR-TKI, CL-387,785, in lung cancer cells (H1975) harboring both L858R activating mutation and T790M secondary mutation in EGFR.

Experimental Design:

CL-387,785 sensitivity and signal transduction in H1975 cells were examined in the presence or absence of HGF or HGF-producing fibroblasts with or without HGF-MET inhibitors.

Results:

HGF induces resistance to CL-387,785 in H1975 cells. Western blotting and small interfering RNA analyses indicated that HGF-induced resistance was mediated by the MET/phosphoinositide 3-kinase/Akt signaling pathway independent of EGFR, ErbB2, ErbB3, and ErbB4. Resistance of H1975 cells to CL-387,785 was also induced by coculture with high-level HGF-producing lung fibroblasts. The resistance was abrogated by treatment with anti-HGF neutralizing antibody, HGF antagonist NK4, or MET-TKI.

Conclusions:

We showed HGF-mediated resistance as a novel mechanism of resistance to irreversible EGFR-TKIs. Our findings provide a novel insight into the involvement of HGF-MET mediated signaling in de novo and acquired resistance to irreversible EGFR-TKIs in lung cancer harboring T790M mutation in EGFR.

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 629.
Abstract 635: Triggering cancer cell death using light-inactivation of the Ki-67 protein
Authors: Rahmanzadeh Ramtin , Rai Prakash , Rizvi Imran , Celli Jonathan , Abu-Yousif Adnan , Baron-Lühr Bettina , Gerdes Johannes , Hasan Tayyaba
Published: February 2011

INTRODUCTION: Specific destruction of cancer cells can reduce the acute systemic toxicity associated with current treatments. This can be achieved by targeting key molecular markers implicated in cancer cell growth. One such marker associated with cell proliferation is the Ki-67 protein (pKi-67) and is highly expressed in cancer cells. However it is a protein that resides inside the nuclei of cells and thus targeting it has been a challenge so far. Here, we show an innovative proof-of-principle approach for efficient killing of proliferating ovarian cancer cells by photoinactivation of pKi-67 with nanotechnology-based photodynamic therapy (PDT). METHODS: Anti pKi-67 antibodies were conjugated to fluorescein isothiocyanate (FITC) which is used as a photoactivatable agent. These conjugates were then encapsulated inside liposomes for intracellular delivery. The efficacy of Ki-67 targeting was evaluated using two in vitro models of ovarian cancer, monolayer cultures and 3D cultures established in our lab. Cell viability after irradiation with laser light at 488 nm was assessed using MTT viability assay for monolayer cultures while a Live/Dead assay kit was used for analyzing 3D cultures. RESULTS: First we demonstrated the delivery of pKi-67 antibodies to the nucleoli of ovarian cancer cells with liposomal constructs. Irradiation of these cells with a 488 nm laser led to a significant loss of viability when pKi-67 was targeted with the TuBB-9-FITC antibodies. The antibody TuBB-9 recognizes a fraction of pKi-67, which is involved in ribosomal RNA synthesis. The efficacy of this approach was also demonstrated in a 3D culture model of OVCAR-5 cells. Incubation of 3D cultures with the liposomal constructs followed by light irradiation led to destruction of their acinar structures over 72 h following treatment. Analysis of cell killing with dependence on acinar size showed that largest acini responded best to the treatment. The specificity of this approach was demonstrated in confluent human lung fibroblasts (MRC-5) where only a small population expresses pKi-67 and consistent with our hypothesis, minimal cell death was observed here. CONCLUSIONS: Our findings suggest that pKi-67 is an attractive therapeutic target in cancer. This approach allows destruction of the most aggressive cancer cells and holds promise as an effective alternative treatment.

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 635.
Abstract 636: Role of Prostaglandin E2 in breast-cancer associated lymphangiogenesis in an in vitro system
Authors: Girish Gannareddy V. , Radan Lida , Tutunea-Fatan Elena , Majumder Mousumi , Bhattacharjee Rabindra , Xin Xiping , Lala Neena , Lala Peeyush K.
Published: February 2011

Lymphatic metastasis is a common occurence in breast cancer. Molecular mechanisms in breast cancer-associated lymphangiogenesis and lymphatic metastasis are poorly defined. We had earlier shown that elevated cyclo-oxygenase (COX)-2 expression by human as well as murine breast cancer cells promotes tumor progression and metastasis by multiple mechanisms: inactivation of host anti-tumor immune cells, stimulation of tumor cell migration and tumor-associated angiogenesis. Furthermore, COX-2 was causally associated with increased VEGF-C expression/secretion in human and murine breast cancer cell lines, thus promoting tumor-associated lymphangiogenesis. VEGF-C production was partially dependent on endogenous PGE-2 mediated activation of EP4 receptors on breast cancer cells thus making EP4 a good therapeutic target. It was unclear whether tumor or host derived PGE-2 had any direct effect on lymphangiogenesis, and if so, whether EP4 receptors on lymphatic endothelial cells played any role. To address these questions, we devised an in vitro lymphangiogenesis assay using a LYVE-1 expressing rat mesenteric lymphatic endothelial cell line (RMLEC) plated on growth factor- reduced Matrigel. Endothelial tube formation by RMLEC was rapidly induced in 12-18 hours after plating on Matrigel even under serum-free conditions, whereas plating them on collagen gel even in the presence of serum did not induce any tube formation. This suggested the presence of some inducing factor(s) in the Matrigel, possibly also present in the ECM in vivo. Matrigel-induced tube formation was completely abrogated in the presence of COX 1/2 inhibitor indomethacin (10mM), COX-2 inhibitor NS-398 (15mM), and a selective EP4 antagonist CJ-042794 (2.5 mM). In each case, an additional presence of PGE2 (1 µM) or an EP4 agonist PGE-1 alcohol (1µM) completely restored the tube formation on matrigel. A similar restoration was also achieved in the presence of serum-free conditioned media (24 h culture) of a COX-2 expressing murine breast cancer cell line C3L5 that induces lymphangiogenesis in vivo. These results indicate the roles of tumor as well as host-derived PGE2 in inducing lymphangiogenesis possibly by activating COX-2/EP4 receptors on lymphatic endothelial cells. Further studies are in progress to identify the tube-inducing component(s) in the Matrigel and C3L5 cell conditioned medium. (Supported by grants from the Canadian Breast Cancer Foundation, Ontario chapter and the Ontario Institute of Cancer Research to PKL. The gift of the RMLEC from Dr Sophia Ran, Southern Illinois Univ School of Medicine is gratefully acknowledged).

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 636.
Abstract 640: SIRT2 deacetylates peroxiredoxin and increases sensitivity to oxidative stress in human breast cancer cells
Authors: CoothanKandaswamy Veena , Chen Jianguang , Joshi Atul , Rao Rekha , Fiskus Warren , Buckley Kathleen M. , Nalluri Srilatha , Bhalla Kapil N.
Published: February 2011

SIRT2, the mammalian ortholog of yeast Hst2, is a predominantly cytosolic and nuclear member of sirtuins, which is the class III, NAD+ dependent, histone deacetylase (HDAC) family. Sirtuins play a key role in modulating cellular stress resistance, longevity and cancer transformation. SIRT2 is known to deacetylate FOXO1 and FOXO3, thereby regulating FOXO-regulated stress resistance. In the cytosol, SIRT2 co-localizes with microtubules and deacetylates α-tubulin at the lysine-40, indicating a role in proper cytokinensis. Here, we determined other biologically important SIRT2 targets and their functions. Knockdown and overexpression of SIRT2 in HEK293 cells significantly altered the acetylation status of many proteins, including a group of proteins ranging in molecular weight from 23-50 kDa. In cells with knockdown of SIRT2, differential in-gel electrophoresis of cytosolic extract of cells followed by mass spectrometric analysis identified several significantly hyper-acetylated proteins, notably peroxiredoxin 1. Peroxiredoxins are ubiquitous family of evolutionarily conserved, thiol-dependent peroxidases, which catalyze the reduction of hydrogen peroxide. SIRT2 co-immunoprecipitated with peroxiredoxin 1, and ectopic overexpression of SIRT2 attenuated acetylation of peroxiredoxin 1 in HEK 293 cells. While acetylation activates, reduced acetylation is known to inhibit the activity of peroxiredoxins. Ectopic overexpression of SIRT2, but not the catalytically dead SIRT2 mutant, markedly increased reactive oxygen species (ROS) levels as well as increased the sensitivity of the human breast cancer MCF7 and MDA MB231 cells to oxidant stress induced by hydrogen peroxide. SIRT2 overexpression in breast cancer cells also increased their sensitivity to ROS generating agents such as arsenic trioxide and menadione. These findings demonstrate that SIRT2 plays an important role in regulating oxidative stress in cells by modulating the activity of peroxiredoxin 1. The findings also highlight the possibility that increased SIRT2 levels and activity can be therapeutically exploited for augmenting the antitumor effects of therapeutic agents that induce cancer cell death by increasing intracellular ROS levels.

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 640.
Abstract 646: Identification of FURIN as a tumor maintenance gene in solid tumors by in vivo Retroviral Mutagenesis and Directed Complementation
Authors: Lorena Lerner , Liu Qing , Yang John , Krieger Brian , Tao Nianjun , Zarycki Jodi , You Fanglei , Kabbarah Omar , Robinson Murray O. , Chiu M. Isabel
Published: February 2011

To identify tumor maintenance genes and pathways, we applied a genome-wide, large-scale functional screening approach using retrovirus-mediated mutagenesis on a number of inducible murine solid tumor models, including a HER2-driven breast tumor model and kRAS-driven breast and lung cancer models, in which viral integration could maintain established tumors in the absence of the initiating oncogene.

We identified more than 3,000 recurrent viral integration sites in a collection of about 400 tumors. Whereas some genes were targeted by viral integration in specific tumor models, others were prominently hit in all the models. For example, insertions targeting the Furin locus were detected in tumors from all three models. In such tumors, viral integrations mapped to the Furin promoter consistent with the notion that activation of this gene and/or pathway may represent a common mechanism of tumor maintenance.

Using our inducible directed complementation (DC) platform, we show that introduction of a wild type FURIN cDNA can maintain primary tumors in vivo in the absence of the initial driving oncogene. The FURIN DC tumors provide a useful pre-clinical model with which we can examine the effects of FURIN antagonists on tumor maintenance. Next, we sought to extend the utility of these validated DC tumor models to drug discovery by establishing FURIN-dependent cell lines for high-throughput screening.

Here we describe detailed molecular characterization of the FURIN DC tumor models as well as from the cell lines derived from these tumor models. Furthermore, FURIN DC Tumor Models as wells as cell lines serve as a unique experimental tools, with which response to novel targeted therapies and other cancer agents can be studied.

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 646.
Abstract 654: High-throughput RNAi screening for the identification of genes that potentiate paclitaxel response in metastatic breast cancer
Authors: Henderson Meredith C. , Gonzales Irma M. , Arora Shilpi , Peralta Lourdes , Little Tanya H. , Choudhary Ashish , Azorsa David O.
Published: February 2011

In the United States, metastatic breast cancer is currently the second leading cause of death in women after lung cancer and it has been estimated that over 400,000 women worldwide die from the disease each year. Two chemotherapeutic agents that have shown much promise in treatment of metastatic breast cancer are paclitaxel and docetaxel, both being members of a class of drugs known as taxanes. Although the current regimens in the treatment of metastatic breast cancer often include one of these taxanes, the overall response rate is between 30-60%, indicating that about half the patients that receive treatment for metastatic breast cancer do not respond. In order to improve paclitaxel response in breast cancer cells, we employed a functional genomics based approach, utilizing high-throughput RNAi (HT-RNAi) to identify genes that, when silenced, would sensitize breast cancer cells to paclitaxel. The HT-RNAi assay was developed to quantify the growth inhibition of MDA-MB-231 breast cancer cells transfected with siRNA and treated with an EC30 dose of paclitaxel. For screening, MDA-MB-231 cells were reverse-transfected with siRNA from a library targeting 7000 druggable genes (2 siRNA per gene). Twenty-four hours after siRNA transfection, an EC30 dose of paclitaxel was added and cell viability was assessed after 72 hours of drug exposure. The data was analysed using the B-score method. Following data analysis, we generated a list of “gene targets”, which upon silencing individually, lead to potentiation of paclitaxel in breast cancer cells. Validation of these genes will identify potential therapeutic targets for novel paclitaxel combinations in order to improve clinical efficacy. Furthermore, these findings demonstrate the effectiveness of using HT-RNAi as a tool for identifying sensitizing targets to chemotherapeutic 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 654.
Abstract 658: Identification of a molecular signature after L-methionine treatment in MCF-7 and LNCaP cancer cells
Authors: Benavides Max A. , Hu Dong , Baraoidan Marie K. , Bruno Annette , Du Pan , Lin Simon , Yang Wancai , Bland Kirby I. , Grizzle William E. , Bosland Maarten C.
Published: February 2011

Our previous research has shown that the essential sulfur-amino acid methionine inhibited proliferation of breast and prostate cancer cell lines, concomitant with p53 modification. The aim of this study was to increment understanding of the mechanisms underlying these effects by determining the influence of L-methionine on functional molecular signatures in these cell lines. Wild-type p53-expressing MCF-7 (breast cancer) and LNCaP (prostate cancer) cells were treated with L-methionine (5mg/ml) for 72hrs. Changes in molecular signatures of these cells were examined by microarray analysis of 15,814 probes in triplicate samples. Genes with significant expression changes were identified by False Discovery Rate analysis (p-value < 0.01; fold-change > 1.5). In LNCaP cells, 325 genes were up-regulated by methionine treatment and 517 genes down-regulated. In MCF-7 cells, 86 genes were up-regulated by methionine and 135 genes up-regulated. Ninety eight genes were regulated in the same direction by methionine in both cell lines; 10 of these genes were up-regulated (AFF3, AKR1C2, ATF3, C8ORF4, GPX8, LAMA3, RBM4B, NQO1, SH3BGRL, and VAMP5) and 88 genes were down-regulated. The expression of five other genes (H1F0, TNF, CENPN, ACAT2, and DHRS2) was changed in opposite directions in the two cell lines. Increases in expression were verified by quantitative RT-PCR. Several of the up-regulated genes encode proteins involved in cellular redox regulation and many of the down-regulated genes belonged to protein kinase families. These observations suggest that methionine is an enhancer of antioxidant activities and has potential as an anti-cancer agent that targets breast and prostate cancer 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 658.
Abstract 666: Regulation of IGF-1 signaling by lysosomal cathepsins in neuroblastoma
Authors: Soori Mehrnoosh , Cartledge Donna , Glazewski Lisa , Lu Guizhen , Mason Robert W.
Published: February 2011

Neuroblastoma is the most common extracranial solid tumor of childhood. Despite intensive combination therapies, the survival rate for children diagnosed with high-risk neuroblastoma remains below 40%, with surviving children suffering from severe side-effects of aggressive surgery, cytotoxic chemotherapy and radiation. We show that specific inhibition of a group of lysosomal proteases results in accumulation of autophagic vacuoles and eventual apoptosis of neuroblastoma cell lines. Cell death is not caused in breast and prostate cancer cell lines or mouse primary fibroblasts, indicating specificity of the apoptotic response in neuroblastoma. In this study we have examined mechanisms by which inhibition of these proteases can lead to the specific death of these tumors. IGF-1 signaling is essential for cellular proliferation, survival, and apoptotic resistance. We found that in neuroblastoma cells IGF-1 signaling is impaired by inhibition of cathepsins B and L. Western blotting shows that lower molecular weight fragments of the IGF-1 receptor beta subunit accumulate in inhibitor-treated cells, indicating that lysosomal proteases are essential for the complete the degradation of the IGF-1 receptor. Phosphorylation and activation of full-length IGF-1 receptor was not modified by inhibitor treatment, but phosphorylation and activation of MAPK and Akt (downstream effectors of IGF-1 receptor signaling) was greatly diminished. Fractionation of cellular lysates showed that the IGF-1R β fragments accumulate in compartments that contain a cleaved form of LC-3, a marker of autophagic vacuoles. Intermediates of the IGF-1 signaling cascade were also sequestered within these compartments, and significantly depleted in the cytoplasm. We conclude that inhibition of lysosomal proteases results in sequestration of critical components of the cell signaling transduction pathway, causing cell death by uncoupling of cell surface and endosomal signaling from intracellular effector signals.

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 666.
Abstract 669: Sunitinib enhances TRAIL-induced apoptosis in preclinical colon cancer models
Authors: Mahalingam Devalingam , Bansal Sanjay , Medina Ernest , Kelly Kevin , Swords Ronan , Cool Robbert , de Jong Steven , Carew Jennifer S. , Giles Francis J. , Nawrocki Steffan T.
Published: February 2011

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may have promise as an anticancer agent due to its selectivity toward tumor cells. However, resistance to TRAIL occurs frequently and is primarily due to defects in apoptosis signaling or activation of survival pathways. Sunitinib is an inhibitor of multiple receptor tyrosine kinases that promote cell survival and drug resistance. We hypothesized that disrupting pro-survival signaling cascades with suntinib would circumvent this mechanism of resistance and strongly enhance TRAIL-mediated apoptosis. We tested this hypothesis by investigating whether inhibition of survival signaling pathways with sunitinib could augment the anticancer activity of TRAIL in preclinical models of colon cancer.

Sunitinib significantly enhanced the anticancer activity of TRAIL in a panel of colon cancer cell lines as evidenced by increased caspase cleavage and DNA fragmentation. The TRAIL/sunitinib combination also promoted enhanced JNK activation and stimulated a decrease in the expression of XIAP, c-IAP2 and Bcl-xL, which correlated with apoptosis. siRNA-mediated knockdown of JNK reduced TRAIL/sunitinib-mediated apoptosis indicating that it is an important mediator of efficacy of this combination. We further investigated this combination regimen in HCT116 and HCT15 xenograft models treated with vehicle, TRAIL, sunitinib, or the combination. The TRAIL/sunitinib combination was very well tolerated and significantly reduced tumor burden in both models compared to either single agent treatment. The reduction in tumor volume correlated with increased apoptosis as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and was associated with decreases in XIAP, c-IAP2 and Bcl-xL expression.

Our collective results suggest that sunitinib enhances TRAIL-mediated apoptosis by increasing JNK activation and downregulating multiple anti-apoptotic proteins. These findings are the first report of the therapeutic potential of the combination of sunitinib and TRAIL in preclinical models of colon 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 669.
Abstract 674: Cooperative induction of apoptosis through p53 signaling and mTOR inhibition in neuroblastoma
Authors: Barbieri Eveline , Chen Zaowen , Ludwig Andrew , Kim Eugene , Patterson Danielle , Sikorski Denae , Shohet Jason
Published: February 2011

The use of MDM2 inhibitors (e.g. Nutlin 3a) to stabilize p53 and induce apoptosis is an attractive therapeutic approach for neuroblastoma which is predominantly p53 wild-type at diagnosis and relapse. We sought to identify additional pathways to potentiate the p53 response. Mammalian target of rapamycin (mTOR) signaling pathway is active in neuroblastoma and mTOR inhibition has antiproliferative effects in vivo in neuroblastoma. It was recently demonstrated that two p53 target genes, Sestrin1 and Sestrin2, inhibit mTOR activity, linking genotoxic stress, p53 and the mTOR signaling pathway. We investigated the role of Sestrin1 and Sestrin2 in p53-mediated apoptosis in neuroblastoma and the interaction of mTOR and p53 pathways after their simultaneous blockade using the mTOR inhibitor, Temsirolimus and the MDM2 inhibitor, Nutlin 3a.

We show here that stabilization and activation of wt-p53 through MDM2 inhibition result in significant and rapid p53-dependent apoptosis. Using microarray analysis in primary tumor line and RT-PCR in several neuroblastoma lines, we demonstrate that the global transcriptional response is p53-dependent and that Sestrin1 and Sestrin2 are significantly up-regulated in response to Nutlin. With MTT and Tunnel assays we demonstrate a p53-dependent synergistic effect of combined Nutlin 3a and Temsirolimus treatment on cell growth and apoptosis. Flow cytometric analysis of the phospho-S6 ribosomal protein demonstrates a profound dephosphorylation of S6 in vitro when low dose Nutlin 3a is combined with Temsirolimus. Additional in vivo studies suggest that mTOR inhibition reduces tumor burden and proliferation of neuroblastoma xenografts in nude mice. We conclude that MDM2 inhibition and p53 driven Sestrin1 and Sestrin2 activation enhance the apoptotic response to mTOR inhibition. Thus the therapeutic strategy of combined MDM2 and mTOR inhibition may represent a clinically potent approach to de novo and relapsed neuroblastoma.

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 674.
Abstract 675: Effect of Progesterone on Antioxidant Activity in Cancer Cells
Authors: Nguyen Huyen , Syed Viqar
Published: February 2011

Progesterone (P4) has been implicated as a protective factor for epithelial ovarian and endometrial cancers, yet little is known about its mechanism of action. We previously reported that pregnancy-equivalent doses of P4 inhibited the growth of normal and malignant human ovarian surface epithelial (HOSE) and endometrial cells. Increased generation of reactive oxygen species (ROS) and an altered redox status have been observed in cancer cells. Here, we investigated if P4-induces cell death by modulating major antioxidant enzymes. The exposure of ovarian and endometrial cancer cell cultures to 10−6 M P4 induced time-dependent increases in early and late apoptotic cells and activation of caspase-3. A general caspase inhibitor Z-VAD effectively blocked the P4- induced cell death in a dose-dependent manner. Expression levels of glutathione peroxidase (GPX1), NAD(P)H:quinine oxidoreductase (NQO1) and copper and zinc SOD (CuZnSOD) were high in cancer cells. Treatment of cancer cells with progesterone at 10−8 M for a period of 72 h induced significant loss of GPX1, NQO1 and CuZnSOD protein expression. The inhibitory action of progesterone was blocked by the specific progesterone antagonist (miferpristone, 10−5 M), confirming P4 specificity. Further investigation on the regulation of antioxidant enzymes by P4 may provide new insights into P4's anticancer activities and facilitate the development of new strategies to use P4 as an anticancer agent.

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 675.