bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2812: mTOR kinase mediates dissemination and colonization of breast cancer metastasis
Authors: Janes Matthew R. , Guo Xin , Elia Marikka , Staunton Jocelyn , Jessen Katti , Rommel Christian , Liu Yi
Published: August 2012

Background: Early events of metastatic dissemination arise from the ability of tumor cells to acquire a number of biological changes which allow for their survival in distal microenvironments. Among the early events that initiate disseminated tumor populations include epithelial-to-mesenchymal transition (EMT) and increased potential for invasion that will ultimately lead to colonization of distal sites. The PI3K/mTOR pathway has been linked to clinic-pathalogical features of invasion and metastasis of multiple tumor types. Results: We sought to investigate using both in vitro and in vivo models of metastasis a panel of PI3K/mTOR selective inhibitors, in which INK128, a selective mTOR kinase inhibitor in clinical development was included. Using live-cell time lapse imaging experiments to monitor cell migration and invasion we found that INK128 significantly inhibited motility and velocity of metastatic breast cancer models in wound assays and decreased the ability of tumor cells to migrate through membranes and matrigel. The ability of INK128 to selectively inhibit tumor cell migration in vitro prompted us to assess whether INK128 can prevent tumor dissemination in vivo. In metastatic breast cancer xenograft models we monitored the growth of tumors implanted in mammary fat pads (MFP) and metastatic dissemination by serial noninvasive whole body bioluminescent imaging. We first assessed and characterized the degree of spontaneous metastasis to the lung, liver, auxillary lymph nodes, chest cavity of mice and subsequently the effect of mTOR kinase inihibition on primary tumor growth on the MFP versus overt metastatic dissemination upon daily, oral treatment with INK128. We show that INK128 blocked drastically metastatic spread in a dose-dependent manner without significantly affecting the growth of primary tumors. Interestingly, we found that INK128 did not affect the ability of breast cancer cells to enter into the lungs following an alternative experimental lung metastasis model by lateral tail vein injection but INK128 greatly diminished the capacity of breast cancer cells to subsequently colonize and expand. Conclusion: Our results position mTOR on a metastasis-sustaining axis that engages downstream pathways to support the biological machinery of migration, invasion, and fitness of initiating breast cancer cells during the establishment of thoracic metastases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2812. doi:1538-7445.AM2012-2812
Abstract 2813: Modulation of Signal Transducer and Activator of Transcription 3 by emodin through the induction of a phosphatase leads to the suppression of HCC growth in orthotopic mouse model
Authors: Shanmugam Muthu K. , Ong Tina H. , Peramaiyan Rajendran , Subramaniam Aruljothi , Hui Kam M. , Sethi Gautam
Published: August 2012

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third cause of global cancer mortality. Activation of signal transducer and activator of transcription 3 (STAT3) is commonly encountered in tumor cells and is a critical mediator of oncogenic signaling in HCC and controls the expression of several genes involved in proliferation, survival, metastasis and angiogenesis. Current drug-targeted therapies, besides being expensive, are associated with serious side effects and morbidity. Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. In this study, we tested an agent, emodin, a quinone, for its ability to suppress STAT3 activation in HCC cells and in nude mice model along with prospectively testing the hypothesis of STAT3 inhibition in a virtual predictive functional proteomics tumor pathway technology platform. We present evidence that emodin can inhibit both constitutive and inducible STAT3 activation in HCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, and Janus-activated kinase-2. Vanadate treatment reversed the emodin induced down-regulation of STAT3 activation, suggesting the involvement of SHP1. Indeed, we found that emodin induced the expression of tyrosine phosphatase SHP1 and deletion of SHP1 by small interfering RNA abolished emodin ability to inhibit STAT3 activation and provide new evidence on the critical role of SHP1 in mechanism of action of emodin. Emodin inhibited proliferation, and produced a synergistic apoptotic effects in the presence of paclitaxel and doxorubicin in HCC cells. When administered i.p., emodin inhibited the growth of human HCC orthotopic tumors in male athymic nu/nu mice. In conclusion, results from experimental and predictive studies suggest that emodin exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling cascade in HCC both in vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2813. doi:1538-7445.AM2012-2813
Abstract 2815: Sylvestrol inhibits protein synthesis by downregulating the AKT/mTOR pathway
Authors: Yang Jilai , Shen Qi , Newman David J. , Kinghorn A. Douglas , Swanson Steven M.
Published: August 2012

Silvestrol, a cyclopenta[b]benzofuran lignan present in the fruits and twigs of the tropical tree Aglaia foveolata Pannell (Meliaceae), is toxic to human cancer cell lines in culture with a potency similar to those of FDA approved anticancer chemotherapeutic agents. We report here that silvestrol has a mean GI50 of approximately 3 nM in the 60 cell-line panel of human cancer cell lines maintained at the National Cancer Institute. Furthermore, silvestrol is at least as effective as FDA-approved anticancer agents in the treatment of human cancer cells in vivo when tested in the hollow fiber assay or the inhibition of human tumor xenograft tumors propagated in immunedeficient mice. Others as well as our group have reported that silvestrol inhibits protein biosynthesis specifically without affecting the cellular production of either RNA or DNA. Pelletier and colleagues have shown that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. In the present study, we test the hypothesis that disruption of protein synthesis by silvestrol can impact cell signaling pathways upstream of translation. Human colon cancer cells designated HT-29 were exposed to silvestrol at concentrations as high as 100 nM for as long as 48 h prior to harvest and evaluation by immunoblot. Protein signaling molecules known to regulate the protein synthesis machinery were evaluated for their abundance and state of phosphorylation as a function of silvestrol exposure. We observed down-regulation of the p110α catalytic subunit of PI3K, which phosphorylates PIP2 to PIP3. PIP3 then recruits PDK1 and AKT to the plasma membrane, where PDK1 can phosphorylate AKT. A decrease in the amount of PDK1 protein and a significant decrease in phosphorylation of AKT at ser 473 occurred. Moreover, a decrease in both the abundance and phosphorylation of mTOR was evident, which is a key component of mTORC2. Consistent with these findings, phosphorylation of AKT at thr 308 was significantly reduced. Finally, silvestrol exposure resulted in a decrease in phosphorylation of the S6 ribosomal protein serine 235/236. These data indicate that silvestrol treatment can disrupt key signaling pathways leading to protein synthesis production in human cancer cells. This work was supported by NIH grants U01 CA52956 and P01 CA125066.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2815. doi:1538-7445.AM2012-2815
Abstract 2820: Vorinostat abrogates ridaforolimus-induced activation of Akt in synovial sarcoma cells: A possible rationale for their synergism
Authors: Morgan Sherif S. , Cranmer Lee D.
Published: August 2012

Background: Curative treatments for patients with metastatic synovial sarcoma (SS) do not exist and such patients have a poor prognosis. Previously, we demonstrated that the combination of the mTOR inhibitor ridaforolimus and the HDAC inhibitor vorinostat exhibited synergism in a variety of cell lines. Here, we explore whether the Akt pathway is potentially involved in mediating the synergistic effects of ridaforolimus and vorinostat. Methods: Two SS cell lines (HS-SY-II and SYO-I) were treated with the single agents or with combinations of ridaforolimus and vorinostat. After 72 hours, cell viability was measured using the cell proliferation assay (MTS). Combination Indices (CI) were calculated to determine whether each combination was synergistic, additive, or antagonistic. Western Blot analysis assessed alterations in total and phospho-Akt protein levels in response to drug treatment. Results: Ridaforolimus IC50 was 10.9nM in HS-SY-II and 23.1nM in SYO-I; vorinostat IC50 was 440nM in HS-SY-II and 561nM in SYO-I. CI were 0.28 and 0.63 in HS-SY-II and SYO-I, respectively, indicating synergism between the two agents. The ridaforolimus/vorinostat combination was assessed in other tumor types, including osteosarcoma (U2OS), metastatic melanoma (Stew1 and Stew2), pancreatic cancer (Panc1 and BxPC3), and lung cancer (A549). The combination was synergistic in all cell lines: CI ranged from 0.37 to 0.77, except in Panc1, where it was additive (CI=0.92). As previously observed with other mTOR inhibitors, ridaforolimus alone increased pAkt-ser473 levels in SS cell lines; this effect was abrogated by the addition of vorinostat. Conclusions: The combination of ridaforolimus and vorinostat demonstrates in vitro synergism in SS as well as in a variety of other tumor types. The addition of vorinostat prevented ridaforolimus-induced Akt activation, a possible mechanism of resistance to mTOR inhibition. Adding HDAC inhibition to mTOR inhibitors may be a route to circumvent Akt-mediated resistance to mTOR inhibitors. Our results also indicate that this combination may demonstrate broad anti-neoplastic activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2820. doi:1538-7445.AM2012-2820
Abstract 2822: Characterization of the effects of PLX4720 in the immunocompetent BRAF/PTEN mouse melanoma model
Authors: Marzuka Alexander , Bosenberg Marcus
Published: August 2012

Vemurafenib is a BRAF inhibitor approved by the FDA for the treatment of BRAF V600E-positive metastatic melanoma after a phase III clinical trial demonstrated improved survival in patients taking vemurafenib compared to dacarbazine. As the first personalized treatment for metastatic melanoma to show a survival benefit, vemurafenib is a major breakthrough in the field. Although tumors in patients frequently shrink significantly within weeks of initiation of treatment, resistance eventually develops in many patients, with a median progression free survival of approximately 7 months. Evaluation of the biomarker and phenotypic correlates of response are difficult to determine as paired pre-treatment and on-treatment biopsies generally only represent one on-treatment time point when available. A better in vivo understanding of the effects of BRAF inhibitor-treatment of relevant mouse models may yield insights into the nature of the clinical response and subsequent relapse. The conditional BRAF/PTEN mouse model of melanoma that we co-developed is ideal to study this process. In this non-xenograft model, expression of BRAF V600E and loss of PTEN is under control of the Cre-Lox recombination system. Expression of the Cre recombinase is under control of the tyrosinase promoter, ensuring that recombination is limited to melanocytes. The Cre recombinase is fused to an estrogen receptor that inhibits the activity of the recombinase. Activation of the Cre recombinase requires topical application of 4-hydroxytamoxifen, allowing for induction of recombination at specific time points in the life span of the mouse. The use of this mouse model allows for more thorough evaluation of the time course and phenotype of BRAF-inhibitor response in melanoma in an immunocompetent host. Specific studies include evaluation of the effect of PLX4720 on the expression, localization, and activation of proteins involved with growth arrest, apoptosis, and glucose uptake.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2822. doi:1538-7445.AM2012-2822
Abstract 2829: Activity of EP-100 in Non-Hodgkin's Lymphoma - synergy in combination
Authors: Leuschner Carola , Coulter Ann , Giardina Cody , Alila Hector W.
Published: August 2012

EP-100 is a targeted anti-cancer peptide comprised of Luteinizing Hormone Releasing Hormone (LHRH) fused to a membrane-disrupting peptide (MDP). It is currently in Phase 1 clinical trial in tumors that over-express LHRH receptors. EP-100 kills cancer cells directly via membrane disruption. We tested a combination of EP-100 and doxorubicin in multi-drug resistant Non-Hodgkin's Lymphoma (NHL) cell lines and primary cells from refractory/relapsed NHL patients. Cells were cultured in the presence of EP-100 (0.00001-100 μM) or unconjugated MDP alone or in combination with doxorubicin (0.0000056 - 56.5 µM) and EP 100 at 0.5, 5, 50 and 500 nM. Cytotoxicity was determined by membrane integrity and cell viability assays. LHRH receptor expression was determined by flow cytometry. The effect of EP-100 on purified human recombinant p-glycoprotein (h-pgp) pump was measured by ATPase activity. The IC50 values [µM] for EP-100 alone were 0.52±0.13, 0.95±0.2, 2.8±0.5, 0.9±0.13 and values for unconjugated MDP were 59±1.5, 25.6±1.7, 6.1±0.8 after 5 h of incubation for Daudi, Raji, Toledo, Hut78 cells, respectively. EP-100 specifically killed NHL patient cells and unconjugated MDP was ineffective. The IC50 values for EP-100 were 1.2 ± 0.1µM for cells obtained from three Mantle Cell Lymphoma patients (N=3), 2.3± 0.1 µM for Diffuse Large B Cell Lymphoma patient (N=1), 1.7± 0.3 µM for Follicular Lymphoma patients (N=4), and 1.6 ± 0.1 µM for one Waldenstr[[Unable to Display Character: ő]]m Macroglobunemia patient. EP-100 or unconjugated MDP did not kill B-cells from normal subjects (N=2) after 5 hour incubation. LHRH receptors were over-expressed on cell lines and patient cells. Combination of EP-100 with doxorubicin resulted in synergistic responses after 72 hours of incubation and Combination Index was <1. EP-100 directly inhibited the h-pgp ATPase activity. These results show that specific killing of NHL cells by EP-100 is mediated via over-expression of LHRH receptors and the synergy between EP-100 and doxorubicin is due to direct inhibition of EP-100 on h-pgp pump.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2829. doi:1538-7445.AM2012-2829
Abstract 283: OOPs: Novel HIF-1α mimics
Authors: Grishagin Ivan V. , Olenyuk Bogdan Z. , Bullock Brooke , Arora Paramjit S.
Published: August 2012

Alpha subunit of the hypoxia-inducible factor 1 (HIF-1α) is an important transcriptional regulator of hypoxia-inducible genes responsible for tumor proliferation and metastasis, and therefore HIF-1α is a valuable therapeutic target. In turn, the interaction between C-terminal activation domain (C-TAD) of HIF-1α and the cysteine-histidine rich domain (CH1) of its coactivator p300 is known to play the key role in the HIF-1 pathway. However, to date very few compounds have been reported to disrupt HIF-1α/p300 complex. We have rationally designed and synthesized non-peptidic mimics of HIF-1α CTAD αA-helix with novel oligooxopiperazine backbone (OOPs). These mimics exhibit relatively low toxicity and substantially downregulate expression levels of selected hypoxia-inducible genes (VEGF, c-MET, LOX, GLUT1, CXCR4) in cellulo in a dose-dependent manner.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 283. doi:1538-7445.AM2012-283
Abstract 2830: Multikinase inhibition by TG02 is therapeutically effective in two forms of breast cancer
Authors: Ortiz-Ruiz M Jesús , Burrows Francis J. , Ré-Louhau M Florencia , Ocaña Alberto , Pandiella Atanasio , Esparís-Ogando Azucena
Published: August 2012

Breast cancer (BC) is a heterogeneous disease in which several distinct genetic and histopathological types have been defined. Triple-negative breast cancer (TNBC) lacking ER/PR and HER-2 is the most aggressive and untreatable form. In the case of HER2+ tumors, and despite the emergence of trastuzumab and lapatinib, in the metastatic setting these tumors remain incurable due to the innate or acquired drug resistance. A number of kinases are implicated in the proliferation and survival of breast cancer cells. Here, we characterize the antitumor activity of the multikinase inhibitor TG02 in TNBC and HER2+ BC. TG02 is a novel drug with a unique kinase inhibitory spectrum, targeting both the cell cycle regulatory CDKs 1/2 and the transcriptional regulators CDKs 7/9, along with the emerging oncogenic MAPK ERK5 and the DNA damage response mediator CDK5, all within a narrow low nanomolar potency range. In panels of breast cancer cell lines, TG02 was potently and consistently antiproliferative at low (100nM) and proapoptotic at moderate (250-1000nM), clinically-achievable concentrations. In TNBC cells, the drug usually induced a G2/M arrest, whereas blockade of cell cycle progression at the G1/S boundary was more common in HER2+ lines. Simultaneous multikinase inhibition was demonstrated in several cellular models, showing that cell cycle arrest induced by inhibition of CDK1 and CDK2 progresses to apoptosis associated with profound depletion of the short-lived survival proteins of the Bcl-2 and IAP families downstream of CDK9-dependent loss of RNA polymerase II-driven transcription. TG02 was synergistic in combination with chemotherapy in TNBC models in vitro and in vivo and with trastuzumab and lapatinib in HER2-overexpressing cell lines. A majority of TNBC and HER2+ lines expressed constitutively active ERK5, which was efficiently inhibited by TG02 and may contribute to the antitumor activity of the drug. A robust DNA damage response including CHK1/2 activation and formation of H2AX foci was induced in TNBC cells, presumably due to inhibition of CDK5 by TG02, so we will also report on the mechanism-based combination of TG02 with PARP inhibitors in this setting. These data support clinical development of this compound for the treatment of TNBC and HER2+ BC, especially in combination with standard of care treatments used in these breast neoplasias.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2830. doi:1538-7445.AM2012-2830
Abstract 2835: Discovery of novel inhibitors for ECT2 as a novel therapeutic strategy for lung cancer
Authors: Beyer Tracey E. , Smith Rachel M. , Song Zuohe , Gokhale Vijay , Moses Sylvestor A. , Meuillet Emmanuelle J.
Published: August 2012

The Epithelial Cell Transforming sequence 2 (ECT2) proto-oncogene is a Guanine Exchange Factor (GEF) for RhoA, Rac1 and Cdc42 and is essential to the regulation of cytokinesis. ECT2 contains the Dbl homology and pleckstrin homology (PH) domains, which are the hallmarks of GEFs. ECT2 is over-expressed in primary non-small cell lung cancer (NSCLC) tumors, and injection of ECT2 transfectants into nude mice efficiently induces tumor formation. High level of ECT2 expression is associated with poor prognosis for patients with NSCLC. Knock down of ECT2 expression by small interfering RNAs effectively suppresses lung cancer cell growth, suggesting a specific role of ECT2 in lung cancer development. Taken together, ECT2 may represent an attractive molecular target for inhibiting lung tumor growth. Our studies are based on the hypothesis that ECT2 plays an important role in lung cancer progression and is a novel target for the development of drugs to treat lung cancer. We have built a model for ECT2 PH domain using protein homology modeling. Docking of new compounds selected from commercial drug-like libraries has led to the identification of novel inhibitors of ECT2 PH domain. We have identified several compounds that bind ECT2 PH domain in the low micromolar range (KD<1μM) as measured using surface plasmon resonance spectroscopy and that inhibit the proliferation of a panel of 23 lung cancer cell lines (EC50<50μM). Importantly, inhibition occurred only in mutant-KRAS cells. Two compounds were shown to inhibit actin polymerization using a phalloidin-rhodamine stain as well as in vitro tumor formation using a colony forming assay and migration using a wound healing assay. These two compounds are being evaluated for their anti-tumor activity against several human lung cancer xenografts in SCID mice. The objectives of our studies are 1) to conduct molecular mechanistic studies of the precise role of ECT2 in the development of lung cancer in vivo animal model of lung cancer; 2) to test the most active compounds in cells in culture to provide a mechanistic rationale for their biological activity and 3) to test the anti-tumor properties of the lead compounds in a in vivo model of lung cancer. The overall goals of our work are to validate ECT2 as a therapeutic target for lung cancer and to develop a novel therapeutic strategy and agents for the improved treatment and the eventual cure of lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2835. doi:1538-7445.AM2012-2835
Abstract 2840: Development of a bioluminescent cell-based bioassay to measure Fc receptor functionality in antibody-dependent cellular cytotoxicity
Authors: Cheng Zhi-jie J. , Chung Shan , Garvin Denise , Moravec Rich , Paguio Aileen , Quarmby Valerie , Fan Frank , Surowy Teresa K.
Published: August 2012

Primary peripheral blood mononuclear cells (PBMCs) are routinely used in traditional bioassays to quantify antibody drug potency in antibody-dependent cellular cytotoxicity (ADCC). These bioassays are labor intensive and have high inherent assay variability. Here, we report the development of a bioluminescent cell-based bioassay which measures activation of effector cells via cross-linking of FcαRIIIA with target cell-bound antibodies. For this, Jurkat T-cell stable cell lines that stably express NFAT-luciferase reporter and human FcαRIIIA were generated to replace primary PBMCs as effector cells in ADCC bioassay. Effector cells were also developed in a frozen, thaw-and-use format, to minimize assay variability due to cell culture and handling. The resultant bioassay using this format demonstrated good assay precision and accuracy in bioassay qualification. Bioassay using the engineered effector cells is robust, specific and is able to quantify the potencies of rituximab and trastuzumab, two monoclonal antibody drugs for cancer. When used to measure effects of Fc glycosylation on effector functions of therapeutic antibodies, a linear correlation was observed between relative antibody activity and the extent of Fc glycosylation. Thus, the bioluminescent cell-based reporter bioassay provides a simple and robust approach to measure potency of therapeutic antibodies in ADCC with high precision.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2840. doi:1538-7445.AM2012-2840
Abstract 2845: Prostaglandin E receptor EP4 antagonist RQ00015986 inhibits breast cancer metastasis and protects natural killer cells from PGE-mediated immune suppression
Authors: Ma Xinrong , Holt Dawn , Kundu Namita , Nonomura Kazuhiko , Take Yukinori , Fulton Amy M.
Published: August 2012

The cyclooxygenase-2 (COX-2) pathway is upregulated in breast and other malignancies. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of G-protein-coupled receptors (EP1-EP4). In breast and colon malignancies, EP4 is a principle mediator of pro-tumorigenic and pro-metastatic activities. Our previous studies demonstrated that targeting EP4 with shRNA or by pharmacologic antagonists of EP4 leads to reduced metastasis in a preclinical model of metastatic breast cancer. These studies and others establish EP4 as a potential therapeutic target, however, there is a paucity of effective EP4 antagonists available for preclinical and clinical investigation. Using a novel orally available EP4 selective antagonist, RQ00015986, we have evaluated efficacy in a syngeneic murine model of metastatic breast cancer. Murine and human breast cancer cells express EP4 and receptor occupation with PGE2 (binds all EP) or with PGE1-OH (EP4 agonist) results in adenyl cyclase activation and elevated intracellular cAMP. RQ00015986, in uM/L concentrations, was able to prevent EP4-mediated adenyl cyclase activation in murine mammary tumor cell lines 66.1 and 410.4. Breast tumor cells migrate in response to either PGE2 or PGE1-OH and RQ00015986 inhibited this response. Pretreatment of 66.1 cells with RQ00015986 prior to i.v. injection into syngeneic mice resulted in 58-69% inhibition in numbers of lung tumor colonies. Natural Killer (NK) cells are critical to the control of metastatic dissemination. The ability of RQ00015986 to inhibit lung tumor colonization was completely lost in hosts depleted of mature NK cells. We have reported previously that NK cell functions in tumor-bearing mice are severely depressed through the actions of tumor-derived PGE2 acting on EP4 receptors expressed on the NK cell. The capacities of NK cells to migrate, to produce cytokines and to lyse mammary tumor target cells are each inhibited through PGE2. In the presence of RQ00015986, the capacity of cultured NK cells to migrate and to produce Interferon-α was restored to normal levels. More interestingly, treatment of tumor-bearing mice with RQ00015986 in vivo also restored NK cell functions. This report provides evidence that a novel EP4 antagonist may have therapeutic potential in cancer and may act by antagonizing EP4 prometastatic functions on the malignant cell as well as protecting NK effector cells from PGE2-mediated immune suppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2845. doi:1538-7445.AM2012-2845
Abstract 2846: Exploring ERBB3 as novel drug target in lung cancer
Authors: Noto Alessia , Aurisicchio Luigi , Vitis Claudia De , Marra Emanuele , Roscilli Giuseppe , Mancini Rita , Ciliberto Gennaro
Published: August 2012

Recent evidences point to ERBB3 as a critical activator of the PI3K/Akt signaling axis and suggest that its trans-phosphorylation is linked to the occurrence of drug resistance in several cancers. Because of the absence of a functional tyrosine kinase, the best approach to target this receptor is through the development of selective monoclonal antibodies. For this purpose, our group has recently generated a set of mouse monoclonals with low nM affinity for human ERBB3. We have started to assess the effect of these antibodies in lung cancer using two systems: a) PC9 cells, known to be highly sensitive to gefitinib (IC50 0,03 uM), for the DelE746A750 in exon 19 of EGFR and its gefitinib-resistant counterpart PC9-ZD (Koizumi et al, 2005 Int J. Cancer 116, 36); b) primary lung adenocarcinoma cells isolated in our laboratory from Malignant Pleural Effusions (MPEs) and capable of efficient tumor propagation in vivo (Mancini et al, 2011, PlosONE 6: e21320). In PC9 cells anti-ERBB3 mabs, A3 and A4, strongly inhibit ligand-induced pHER3 and pAKT and induce receptor downmodulation. Furthermore, A3 and A4 potentiate the antiproliferative effect of gefitinib in a clonogenic assay, induce G0/G1 arrest, and increase the percentage of cells that undergo apoptosis. In the human primary MPE-derived lung adenocarcinoma culture PEd10, we observed that the expression of ERBB3 and its ligand are upregulated when cells are grown in spheroid vs adherent conditions. In this system, A3 and A4 antibodies significantly downmodulate spheroid propagation. Studies are ongoing to provide further insights as to the mechanism of action of anti-ERBB3 antibodies and to confirm efficacy in in vivo tumor models. Our results suggest that in a subset of lung adenocarcinomas which express high levels of ERBB3, this receptor contributes to cell growth and propagation and that targeting ERBB3 with antibodies capable of inducing receptor downmodulation can be considered a promising therapeutic approach.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2846. doi:1538-7445.AM2012-2846
Abstract 2848: Preclinical activity of OM-RCA-01, a humanized anti-FGFR1 antibody, in renal cell carcinoma (RCC)
Authors: Tsimafeyeu Ilya V. , Zaveleva Elina , Low Walter
Published: August 2012

Background: Fibroblast growth factor (FGF) receptor 1 (FGFR1) is a potential therapeutic target for treatment of metastatic RCC. Early we reported that anti-FGFR1 monoclonal antibody inhibited ligand-induced phosphorylation of FGFR1, activation of MAP kinase and Akt downstream signaling pathways in FGFR1 expressing endothelial (bovine adrenal cortex capillary endothelial cells) and human RCC (Caki-1) lines (Tsimafeyeu et al. AACR/JCA joint conference 2010). Today we investigated the preclinical activity of OM-RCA-01, a novel therapeutic humanized anti-FGFR1 antibody with high affinity (Kd of 1.59 nM), in RCC. Methods: To assess the effect of anti-FGFR1 antibody on FGF-mediated signaling, the human renal carcinoma Caki-1 FGFR1-expressing cells were incubated (0.5% FBS) and were dosed with OM-RCA-01 at 100, 10, and 1 mcg/ml. Control wells were left untreated. Three hours after dosing, basic FGF was added at a concentration of 50 ng/ml. Additional control wells were treated with OM-RCA-01 without FGF-stimulation. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Charles River female NCr nu/nu mice (6-12 weeks of age) were set up with 1 mm3 Caki-1 tumor fragments sc in flank. Tumor sizes were measured in a blind fashion twice a week with a vernier caliper. Mice with established tumors (an average size of 80 - 120 mg) were randomly divided into vehicle, non-specific IgG or OM-RCA-01 groups per 10 animals in group. Animals were treated with antibody using different doses (1 or 10 mg/kg). Endpoint was significant differences in tumor growth delay. Results: In vitro study showed that basic FGF significantly increased proliferation of the human FGFR1-expressing renal carcinoma cells (P=0.011). No effect of basic FGF on cell line proliferation was observed when the cells were incubated with OM-RCA-01 antibody at any concentrations up to 100 mcg/kg in comparison with FGF-untreated control (P=0.855). In vivo, the tumors in untreated mice or mice treated with non-specific IgG continued their aggressive growth to reach the size of 2000 cm3, at which point the mice were killed. In contrast, treatment with OM-RCA-01 not only significant arrested further growth of the tumors (P=0.006) but also demonstrated differences in tumor volume compared with vehicle already on Day 13. A similar anti-tumor activity of OM-RCA-01 was observed when the antibody was given in low (1 mg/kg) or high (10 mg/kg) doses (P=0.917). Conclusions: High-affinity humanized anti-FGFR1 antibody OM-RCA-01 suppressed ligand-induced proliferation of human RCC cells in vitro. OM-RCA-01 has significant early anti-tumor efficacy in Caki-1 xenograft model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2848. doi:1538-7445.AM2012-2848
Abstract 2849: Optimization and pharmacokinetic characterization of potent CXCR4 antagonist CovX-Bodies
Authors: Coronella Julia A. , Fu Yanwen , Johnson Kim , Sakamuri Sukumar , Rizzo John , Willhite Dorian , Rosario Joselyn Del , Doppalapudi Venkata , Bhat Abhijit , Levin Nancy
Published: August 2012

Here we describe a series of CXCR4 antagonist CovX-Bodies, produced by covalently attaching CXCR4 antagonist peptides to a humanized IgG1 molecule with the goal of producing molecules with the potency and specificity of peptides and the pharmacokinetic parameters of an antibody. Extensive SAR was performed on peptides derived from T140, a highly charged 14 amino acid cyclic peptide containing 4 Arginine residues. Both the free peptides and the associated CovX-bodies were potent antagonists of CXCR4 in vitro, and SAR studies were consistent with a model of both allosteric and orthosteric inhibition. However, in vivo pharmacokinetic (PK) studies revealed an unexpected and CXCR4-independent disappearance of the CovX-Bodies from circulation, such that approximately 10% of the injected CovX-Body remained in circulation 1 hour after i.v. injection into mice. Based on the known nonspecific cell-penetrating properties of Arginine-rich peptides, we hypothesized that Arginine replacement and charge neutralization would improve the pharmacokinetics of the CovX-Bodies. We identified substitutions that could replace up to 3 of the 4 Arg residues while retaining potent CXCR4 binding and antagonism, but Arg2 was obligate. However, these CovX-Bodies were only modestly improved in PK, indicating that that the anomalous in vivo behavior was not solely a function of charge or the net number of Arg residues. Only the replacement of Arg2 significantly improved the pharmacokinetics of the CovX-Bodies, but also resulted in a loss of CXCR4 antagonism. Extensive SAR failed to identify a suitable replacement residue for Arg2, nor did alternative structural forms such as 5 member, bicyclic, or reversed ring molecules have significantly improved pharmacokinetics. Based on these data, we conclude that the specific property of the peptide that allows potent inhibition of CXCR4, of which Arg2 is an integral component, also mediates binding to moieties other than CXCR4, leading to the depletion of these molecules from circulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2849. doi:1538-7445.AM2012-2849
Abstract 2850: Demonstration of anti-tumor efficacy in multiple preclinical cancer models using a novel peptide inhibitor (Aurigene-012) of the PD1 signaling pathway
Authors: Sasikumar Pottayil G. , Satyam Leena K. , Shrimali Rajeev K. , Subbarao Krishnaprasad , Ramachandra Raghuveer , Vadlamani Sureshkumar , Reddy Amarnadh , Kumar Aswani , Srinivas Adurthi , Reddy Subba , Gopinath Sreevalsam , Samiulla Dodderi S. , Ramachandra Murali
Published: August 2012

Programmed cell death-1 (PD-1), an immunoreceptor belonging to the CD28 family, plays an important role in negatively regulating immune responses. Blocking of PD-1 signalling pathway has been shown to result in restoration of defective immune cell functions in cancer and chronic infections. PD-1 targeted therapies in the ongoing clinical trials are based on either antibodies or fusion proteins. To exploit unique advantages of peptides over antibodies or fusion proteins towards addressing the limitations of the current clinical candidates, herein we report a peptide based strategy to block the PD-1 signaling pathway. Sequences critical for ligand-receptor interaction were identified and combined in a non-linear fashion. The strategy resulted in a novel peptide, AUR-012 (29-mer), which displayed sub-nanomolar potency in disruption of PD1-PDL1/2 interaction, and highly effective restoration of proliferation and effector functions of splenocytes and PBMCs. In vivo studies demonstrated an excellent PK-PD correlation with sustained PD for >24 h. In preclinical models of melanoma, breast and kidney cancers, AUR-012 showed superior efficacy compared to therapeutic agents currently used in the clinic in inhibition of both primary tumor growth and metastasis. Interestingly, dosing once in three days was equally efficacious as once a day dosing with no signs of overt toxicity and generation of neutralizing activity. These findings support further development of AUR-012 for potential clinical use.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2850. doi:1538-7445.AM2012-2850
Abstract 2852: Combination therapy: Galectin-3C and bortezomib potent anticancer activity and inhibition of angiogenesis, chemotaxis, and NF-kB activation in human multiple myeloma
Authors: Yu Yuefei , Mirandola Leonardo , Jenkins Marjorie R. , Cobos Everardo , Chiriva-Internati Maurizio , Chui Kitty , John Constance M.
Published: August 2012

Introduction Galectin-3 is a human lectin involved in cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Methods We analyzed 9 human MM cell lines tested in vitro for cell proliferation/viability; chemotaxis. The human MM cell lines used in this study were RPMI-8226, U266 (American Type Culture Collection, VA, USA), ARP-1, ARK-B, (gifts from J. Epstein, University of Arkansas for Medical Sciences, Little Rock, AR, USA). The 8226/Dox and 8226/LR-5 cell lines were kindly provided by Dr. William S. Dalton of the H. Lee Moffitt Cancer Center and Research Institute in Tampa, Florida. All MM cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37 °C. The 8226/Dox cells were cultured with 40 nM doxorubicin (Sigma-Aldrich, St. Louis, MO, USA). The 8226/LR-5 cells were cultured in media containing 5 µM mephalan (Sigma-Aldrich). 8226/Dox and 8226/LR-5 cells were maintained in drug-free medium for 1 week prior to drug sensitivity assays. Human umbilical cord vascular endothelial cells (HUVEC, American Type Culture Collection, VA, USA) were maintained in EGM-2 medium (Lonza, Houston, TX, USA) and were used within 10 passages. Furthermore we used U266 MM model in the NOD/SCID mice to evaluate the efficacy of galectin-3C in single or in combination with BOR. Results Galectin-3C exhibited modest anti-proliferative effects on MM cells in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. Bortezomib, a proteasome inhibitor approved by the F.D.A. for MM treatment, also inhibited the growth of the U266 tumors. Treatment with both agents was more effective than either alone, such that the average tumor volume of the combination group was just 14.0% of controls at day 35. We show that mechanisms by which galectin-3C facilitates the anticancer activity of BOR include inhibition of angiogenesis, chemotaxis, and NF-kB activation. Conclusion In conclusion, our study shows that in a mouse model of human MM, treatment with galectin-3C alone is efficacious, and that it enhances the anti-tumor activity of bortezomib. These findings provide the proof-of-principle to test galectin-3C clinically for human MM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2852. doi:1538-7445.AM2012-2852
Abstract 2853: Discovery of phosphatases that regulate growth and tumorigenicity of ER-negative breast cancers
Authors: den Hollander Petra , Mazumdar Abhijit , Tsimelzon Anna , Hilsenbeck Susan , Chang Jenny , Mills Gordon , Brown Powel
Published: August 2012

Background: As the leading cause of cancer-related death in women, there is an urgent need to identify therapeutic means for breast cancer in women. 60-70% of breast cancers express the estrogen receptor (ER) and respond to therapies targeting this receptor. The remaining 30-40% of breast cancers that do not express this receptor are cannot be treated with these therapies. To identify novel targets for treatment, we are investigating growth-regulatory pathways in ER-negative breast cancers. The goal is to identify phosphatases that are critical for the growth and tumorigenicity of ER-negative breast cancer. Methods: To identify signaling pathways important in ER-negative breast cancer, we used Affymetrix microarray data obtained from profiling 221 ER-negative and 56 ER-positive breast cancers from the Baylor College of Medicine Tumor Bank. We then performed supervised cluster analysis to identify phosphatases that were differentially expressed in ER-negative and ER-positive breast cancers. This analysis identified 20 phosphatases upregulated (>1.5-fold; FDR=0.05), and 29 phosphatases down-regulated (< 0.66-fold, FDR=0.05) in ER-negative breast cancers as compared to ER-positive breast cancers. Specific siRNA were used to knock-down each of the 20 highly expressed phosphatases in 4 ER-negative, 4 ER-positive breast cancer cell lines, HMECs immortalized with TERT, and HMECs. Cell growth was measured by cell count, and anchorage-independent growth was tested with the soft agar assay. Colonies were counted after 14 days with the Gelcount software. Results: Of the 20 overexpressed phosphatases, 9 showed significant growth reduction after treatment with siRNA in at least 2 of the 4 ER-negative breast cancer cell lines. These 9 phosphatases are now being tested in anchorage-independent growth assays. Based on our results, 3 phosphatases (ACP1, PTP4A3, and CDC25B) have been selected for further study. These are now being tested for their transforming capability in in vitro and in vivo transformation and tumorigenicity assays. Conclusion: We identified a set of phosphatases highly over-expressed in ER-negative breast cancer, and showed that a sub-set of these phosphatases are critical for anchorage-dependent and -independent breast cancer cell growth. These studies have identified critical growth-regulatory phosphatases in ER-negative breast tumors that may be novel targets for the treatment of ER-negative breast cancer. Supported by Komen Promise grant KG081694 (PHB) & Komen SAB grant SAB08-00006 (PHB).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2853. doi:1538-7445.AM2012-2853
Abstract 286: Development and evaluation of a molecular decoy against SLUG function in the breast cancer cells
Authors: Mittal Mukul K. , Chaudhuri Gautam
Published: August 2012

SLUG, also known as SNAI2, is a member of the SNAI superfamily of zinc-finger transcriptional repressors. It is a C2H2-type zinc finger transcription factor that binds to E2-box motif (5′-CAGGTG-3′/5′-CACCTG-3′), and silences gene expression by chromatin remodeling. More than ninety genes are shown to bind tightly to SLUG as was revealed by ChIP-on-chip analysis, showing the large regulatory network involving SLUG. It is recently implicated as the major mediator of the master transcriptional regulator protein TWIST. SLUG represses several genes to directly contribute towards the metastatic transformation of several epithelial cancer cells. Specific inhibitor against SLUG is not known. We describe here the development and evaluation of a nuclease resistant double-stranded DNA decoy that specifically inhibit the repressor activity of SLUG in the human breast cancer cells. The decoy is designed from the VDR gene promoter which is repressed by SLUG. This decoy has two SLUG-binding E2-box sequences. EMSA analysis showed in vitro binding of SLUG to the wild-type decoy but not to the E2-box mutated decoy. In vitro pull down analysis also showed high affinity binding of SLUG to the decoy. Biotin-tagged wild-type decoy DNA could immunopull down SLUG but little SNAIL from the extracts from decoy-transfected cells. The decoy increased the mRNA and protein levels of several SLUG target genes in MDA-MB-231 cells. The decoy also alleviated the repressive activity of SLUG on the SLUG target gene promoters in MDA-MB-231 cells. Since SLUG overexpression is implicated in the drug-resistance and antiapoptosis development in breast cancer cells, the SLUG decoy we developed should be useful to combat breast cancer progression when used with classical anticancer drugs. Supported in parts by the DOD-CDMRP grants BC050641, BC086542 and BC103645 to GC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 286. doi:1538-7445.AM2012-286