bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3615: Erlotinib in combination with different chemotherapeutics is active in a SCID mouse/ human melanoma model
Authors: Schicher Nikolaus , Pehamberger Hubert , Hoeller Christoph
Published: February 2011

Erlotinib is a novel EGF-receptor targeting drug which has been tested in various cancers for its activity, has shown impressive results in non small cell lung cancer, but has so far failed as a single agent in other cancer types. We and others have recently shown that the combination of erlotinib with e.g. the monoclonal VEGF antibody bevacizumab or chemotherapy is more powerful in inducing apoptosis and reducing proliferation or angiogenesis. Due to the fact that erlotinib induces accumulation of tumor cells in the G1-phase of the cell cycle this leads to the assumption that erlotinib prior to or concomitant with chemotherapy might negatively interact with it. The aim of our study was to test different dosing schedules of erlotinib in combination with dacarbazine, paclitaxel or cisplatin in a SCID mouse/ human melanoma xenotransplantation model with 518 A2 and M24 met cell lines. We hypothesize that pausing erlotinib before chemotherapy leads to greater apoptosis.

Six to eight week old female SCID mice were injected with 518 A2 or M24 met melanoma cells and treated either with erlotinib, dacarbazine, paclitaxel or cisplatin as single agents or a combination of erlotinib with either chemotherapy. Groups were divided in control, erlotinib, chemotherapy, erlotinib concurrently with chemotherapy or with a break of 5 days prior to chemotherapy.

While the cytotoxic drugs did show single agent activity in the model system used, the combination with erlotinib exhibited a higher reduction in tumor volume. These effects were substantiated by immunohistochemical analysis where we observed a reduction of angiogenesis and proliferation as well as an increase in apoptosis in the combined treatment arms as assessed with ki-67, CD31 and cleaeved caspase 3 staining, respectively. Interestingly, withholding erlotinib before treatment did not alter the activity of dacarbazine, thereby excluding an inhibitory effect of EGF-receptor inhibition under these conditions.

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 3615.
Abstract 3616: Changes in signaling pathways induced by vandetanib in a human medullary thyroid carcinoma model as detected by reverse phase protein arrays
Authors: Broutin Sophie , Commo Frederic , Hego Caroline , Marty Bérengère , Dupuy Corinne , Ryan Anderson Joseph , Schlumberger Martin , Bidart Jean-Michel
Published: February 2011

Introduction: Medullary thyroid carcinomas (MTC) frequently involve activating mutations in the proto-oncogene tyrosine-kinase receptor RET. Vandetanib (ZD6474, ZACTIMA) is a potent inhibitor of VEGFR-2, EGFR and RET tyrosine-kinase activities which is currently in phase III clinical trial in MTC. The aim of this study was to investigate in vitro and in vivo molecular effects of vandetanib on MTC signaling pathways using a proteomic approach based on reverse-phase protein arrays (RPPA).

Methods: The human MTC TT cell line, bearing a RETC634W activating mutation, was cultured in the absence or the presence of vandetanib (100, 500, 1000 nM) for 4, 24 and 72 hours. MTC TT xenografted athymic nu/nu mice received vandetanib (50 mg/kg/day p.o.) or placebo and xenografts were removed after 1 and 4 days of treatment. Twenty-eight protein extracts from in vitro (n=24) and in vivo (n=4) experiments were profiled by RPPA analysis. Equal amounts of lysates were printed in serial dilutions onto nitrocellulose-coated glass slides. Each slide was then probed with each of a 20-antibodies set, specific for oncogenic pathways (PI3K/AKT/mTOR, MEK/p44-42, STAT3, NFKB) and cell cycle/apoptotic proteins (CDK1, PARP, cleaved-PARP).

Results: In in vitro experiments, vandetanib treatment induced an increase of cleaved-PARP (p<10-5) with a 3-fold rise after 72 hours at 100 nM. Phosphorylation of NFKB was not significantly modified when compared to actin. In contrast, phosphorylation of STAT3 significantly decreased after 72 hours of vandetanib treatment. Overall, the MAPK signaling pathway was dramatically inhibited by vandetanib, as substantiated by the decrease of MEK and p44-42 phosphorylation when compared to their total protein forms (p<10-3). Inhibition of p44/42 and STAT3 phosphorylation were also observed in MTC TT xenografted mice treated by vandetanib. Several proteins were re-analyzed by western-blot analysis and the results were in agreement with those observed in RPPA.

Conclusions: Together these observations demonstrated that, at the protein level, vandetanib induces apoptosis and inhibits MAPK and STAT3 pathways in MTC. Furthermore, our experimental approach confirmed the utility of protein arrays for analyzing changes in signaling pathways induced by tyrosine-kinase inhibitors.

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 3616.
Abstract 3620: Efficacy of Vandetanib on EGFR mutant lung cancer cells with PTEN loss
Authors: Takeda Hiromasa , Ohashi Kadoaki , Kataoka Itaru , Ichihara Eiki , Takigawa Nagio , Tanimoto Mitsune , Kiura Katsuyuki
Published: February 2011

Introduction

EGFR gene is frequently mutated in non-small cell lung cancer (NSCLC) of never smokers, especially in Asian ethnicity. The mutations often predict dramatic response to EGFR-tyrosine kinase inhibitors (TKIs) like gefitinib. PTEN is a tumor-suppressor gene deactivating PI3K in downstream of EGFR signaling. Approximately 2% to 9% in NSCLC tumors were considered to be PTEN loss. PTEN loss and EGFR mutation co-occurred in 1 out of 24 EGFR mutant patients with lung adenocarcinoma (Sos, et al. Cancer Res 69, 2009). PTEN loss was considered to represent primary or acquired resistance to EGFR-TKIs. Therefore, a new strategy is needed to break through for the resistance due to PTEN loss.

Objective

We clarify the effectiveness of vandetanib, EGFR and vascular endothelial growth factor receptor (VEGFR) inhibitor, against lung cancer cell lines harboring EGFR mutations with PTEN loss.

Materials and methods:

Two EGFR mutant (deletion in exon19) NSCLC cell lines, PC-9 (PTEN wild type) and NCI-H1650 (PTEN loss) were used. Other two cell lines with PTEN loss, prostate cancer cell line (PC-3) and glioblastoma cell line (U-87MG) were also examined. We transfected intact PTEN gene into H1650 cells and knocked down PTEN expression in the PC-9 cells using shRNA. Antiproliferative activity was determined by MTT assay in terms of 50% inhibitory concentration (IC50) values. We also examined the effectiveness of the drugs (15 mg/kg/day) in xenograft model. Protein expression profile was examined by Western blotting. Expression of VEGFR mRNA levels was also assessed by RT-PCR.

Results

PC-9 cells were more resistant to vandetanib (IC50: 0.08 μM) than to gefitinib (IC50: 0.01 μM). However, mean IC50 values of vandetenib were significantly lower than those of gefitinib against H1650 cells (2 μM vs 25 μM), PC-3 cells (8 μM vs 25 μM), and U87MG cells (6 μM vs 28 μM) with PTEN loss. EGFR downstream signals including pAKT, pMAPK, and pSTAT3, and VEGFR were not different in the H1650 cells treated with vandetanib or gefitinib. In the xenograft model using H1650 cells, vandetanib was also more effective than gefitinib. Although PTEN transfected H1650 cells did not restore sensitiveness to gefitinib in vitro, the xenograft tumors responded to gefitinib as well as vandetanib. Knockdown of PTEN lead six times more resistance to gefitinib in PC-9 cells.

Conclusion

PTEN loss may play a role to determine the sensitivity to gefitinib in NSCLC with EGFR mutations. Vandetanib is more effective in NSCLC with EGFR mutation and PTEN loss.

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 3620.
Abstract 3621: The multi-tyrosine kinase inhibitor Sunitinib reduces prostate cancer growth through autocrine and paracrine mechanisms
Authors: NGUEWA PAUL A. , Díaz Roque Díaz , Redrado Miriam , Collantes María , Peñuelas Ivan , Saenz Irene Manrique , Serrano Diego , Agliano Alice , Calvo Alfonso
Published: February 2011

Prostate cancer (PrCa) is the most common malignancy and the second leading cause of cancer death in men. Although localized PrCa can be successfully cured, advanced hormone refractory tumors are resistant to current therapeutic regimes. Therefore, novel approaches for effective malignancy control are still required. Molecular targeted therapies have emerged as promising alternatives to classical chemo/radiotherapy. The clinical benefit of the multi-tyrosine kinase inhibitor Sunitinib, which blocks the activity of receptors such as PDGFR, VEGFR1/2, FLT3, and c-KIT has been proven in renal carcinoma, GIST, and neuroblastoma. In this work, we have tested the therapeutical efficacy of sunitinib against PrCa both in vitro and in vivo, using the androgen-independent cell line PC-3. These cells express target receptors PDGFRa/b, and VEGFR1/2. Sunitinib dramatically reduced cell proliferation in vitro (P<0.0001) in a dose-dependent manner (with an IC50 of 1-2 micro-M) and colony formation (P<0.0001), through an autocrine mechanism. Moreover, when combined with doses of 2 and 4 Gy ionizing radiation, sunitinib enhanced the inhibitory activity of radiotherapy in colony formation assays. In vivo experiments revealed that PC3 tumor-bearing mice treated with 80 mg/Kg/day sunitinib had significantly smaller tumors (63% reduction) than controls. A single dose of 6 Gy caused tumor shrinkage by 80%, whereas the combination of both therapies resulted in more than 90% tumor reduction, suggesting that sunitinib could be used as a radiosensitizer. Positron emission tomography (PET) analyses with 18-FDG, FMISO, and FLT tracers showed a decrease in glucose uptake, hypoxia, and cell proliferation, respectively, in sunitinib-treated mice, as compared to controls. Immunohistochemical analysis for CD-31 revealed an expected reduction in tumor angiogenesis in sunitinib-treated tumors in comparison with untreated tumors. Overall, these data support the use of sunitinib as a clinically relevant drug for the treatment of PrCa.

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 3621.
Abstract 3622: Preclinical characterization of EMD1214063, a potent and highly selective inhibitor of the c-Met kinase in Phase I clinical trials
Authors: Bladt Friedhelm , Blaukat Andree , Dorsch Dieter , Fittschen Claus , Friese-Hamim Manja , Graedler Ulrich , Meyring Michael , Rautenberg Wilfried , Schadt Oliver , Stieber Frank
Published: February 2011

c-Met, the receptor for hepatocyte growth factor (HGF), is a well characterized receptor tyrosine kinase, which is crucial for cell functions, such as survival, proliferation, motility and migration. Activating point mutations of c-Met have been identified in human malignancies, including renal carcinoma and lung cancer and constitutive activation of the HGF/c-Met pathway leads to tumor development and progression in cancer animal models. In the light of these findings, c-Met has become a key target for oncology therapeutics.

In order to develop c-Met specific inhibitors, we have conducted a biochemical HTS run with the recombinant kinase domain of c-Met. This approach has enabled us to identify a new class of c-Met inhibitors. Our report will focus on pharmacological and pre-clinical data, characterising EMD1214063, a promising therapeutic candidate currently under clinical testing.

Inhibition of the c-Met kinase by EMD1214063 was potent and highly selective. In in vitro kinase reactions and binding assays, EMD1214063 at a dose of 1μM, inhibited only 5 additional kinases in a panel of more than 250 different potential off-targets more than 50%. In vitro, EMD1214063 interfered with survival, anchorage-independent growth and HGF-induced migration of tumor cells, by blocking HGF-dependent as well as constitutive phosphorylation of c-Met.

In vivo, oral administration of EMD1214063 resulted in a strong inhibition - ranging from delayed growth to complete regression - of HGF-dependent and -independent c-Met driven tumor xenografts. Such anti-tumor activity was observed at a broad dose range, suggesting that EMD1214063 has a wide therapeutic index. The inhibitor was well tolerated, as indicated by the lack of significant weight loss in treated mice. The mechanism of action of EMD1214063 was investigated in vivo in a series of PK/PD studies. Administration of a single dose of EMD1214063 induced a complete and long-lasting inhibition of c-Met phosphorylation in the tumor xenografts. Furthermore, it strongly reduced the plasma levels of tumor-derived IL-8. c-Met phosphorylation and plasma IL-8 levels decreased as a function of the drug concentration in the tumor and plasma.

Taken together, the high c-Met selectivity, potency and in vivo efficacy of EMD1214063 render this compound a very promising candidate for the treatment of patients bearing c-Met-driven tumors.

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 3622.
Abstract 3626: Development of inhibitors of the fibroblast growth factor receptor (FGFR) kinase using a fragment based approach
Authors: Squires Matthew S. , Perera Timothy , Saxty Gordon , Murray Chris , King Peter , Ward George , Feltell Ruth , Rich Sharna , Angibaud Patrik , Lewis Edward J. , Gilissen Ron , Harada Isobel , Fazal Lynsey , Irving Julie A. , Batey Mike A. , Zhao Yan , Newell David R. , Thompson Neil T.
Published: February 2011

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. FGFR is a receptor tyrosine kinase which activates the extracellular signal-regulated kinase / mitogen-activated protein kinase and the protein kinase B / Akt pathways which promote cell growth and survival. Amplification, over-expression or activating mutations of fibroblast growth factor receptors have been associated with bladder tumours, multiple myeloma, hormone-refractory prostate cancer and breast cancer.

Multiple lead series of FGFR inhibitors were developed using Astex's fragment based medicinal chemistry approach, Pyramid™, linked to high throughput X-ray Crystallography. We describe here the characterisation of some examples of these lead molecules. In particular we detail the pharmacological profile of a compound from one of these lead series that demonstrated activity against FGFR 1-4 with an IC50 <100nM in an isolated kinase assay. This compound inhibited FGFR1-4 kinase activity in BaF3 cell lines engineered to express the relevant kinase fusion proteins and proliferation and survival of a panel of FGFR-dependent human tumour cell lines derived from several different tissues. The cytotoxic activity was >10 fold lower in cell lines lacking FGFR expression. We demonstrate inhibition of FGFR 2 and 3 phosphorylation in gastric and multiple myeloma cell lines respectively with associated inhibition of downstream signalling pathways.

This lead molecule has an excellent pharmacokinetic profile and high oral bioavailibility in mice and rats. In xenograft models in mice where aberrant FGF signalling underlies tumour pathology, tumour growth inhibition is observed at doses of 100mg/kg /day orally for 21 days. This xenograft efficacy was observed in several models, with significantly lower activity in models where aberrant FGF signalling is not involved in tumour pathology. This suggests that the mechanism of action is consistent with FGFR inhibition. The pharmacological profile in these models is also distinct from other broader spectrum receptor tyrosine kinase inhibitors.

The pre-clinical data shown here suggests that such compounds warrant further investigation pre-clinically and may benefit patients whose disease is driven by FGFR activity.

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 3626.
Abstract 3634: Overcoming drug resistance to EGFR-tyrosine kinase inhibitors by FUS1 or FHIT-gene therapy in human lung cancer
Authors: Sakai Ryo , Xu Kai , Fang Bingliang , Roth Jack A. , Ji Lin
Published: February 2011

Dysregulation of the epidermal growth factor receptor (EGFR) and apoptotic pathways is a hallmark of human lung cancer and regulate tumor cell proliferation, survival, angiogenesis, and metastasis. Inhibition of oncogenic EGFR activity is proving to be an effective therapeutic strategy for lung cancer. Though EGFR-tyrosine kinase inhibitors (TKIs) are promising drugs, resistance occurs frequently. ZD6474 is an orally available small-molecule that inhibits both EGFR and VEGFR2 tyrosine kinases. FUS1 and FHIT are novel tumor suppressor genes identified in the human chromosome 3p21.3 and 3p14.2 regions, respectively and have been shown to suppress tumor cell growth by induction of apoptosis and down-regulation of multiple oncogenic tyrosine kinase activities in human non-small-cell lung cancer (NSCLC) cells in vitro and in vivo. We evaluated the therapeutic effects of EGFR-TKIs at dose levels equivalent to those of steady-state serum levels achieved in the clinic as single agents and in combination with FUS1- or FHIT-nanoparticle-mediated gene therapy in NSCLC cells. We found that expression of wild type FUS1 or FHIT sensitized NSCLC cells to ZD6474 treatment (at 2µM), as shown by significantly enhanced inhibition of tumor cell growth in a cell proliferation assay. A combination treatment with FUS1-nanoparticles and ZD6474 also significantly enhanced inhibition on tumor cell-induced colony formation in H322 cells compared to treatment with either agent alone. Combination treatment with ZD6474 and FUS1 or FHIT-nanoparticles increased down-regulation of the EGFR signaling pathway and up-regulation of the apoptosis pathway as indicated by the reduced expression of the phosphorylated Akt and ERK1/2 and by enhanced expression of cleaved PARP on Western blots. We found that the expression level of p-Met, which is one of acquired resistant factors to EGFR-TKIs, was reduced by FHIT-gene therapy in H1975 cells. We evaluated the antitumor effects of combination therapy using a fluorometric migration assay and found that the migration of H1299-GFP cells were inhibited by co-treated with ZD6474 and FHIT-gene therapy. Our findings imply that a combination treatment with FUS1 or FHIT-gene therapy and ZD6474 may be a useful strategy for enhancing lung cancer therapy. (supported by NIH Lung SPORE P50CA70907 and RO1CA116322)

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 3634.
Abstract 364: Coat color mutations alter angiogenic responsiveness
Authors: Rogers Michael S. , Birsner Amy E. , D'Amato Robert J.
Published: February 2011

Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependant diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that control angiogenic responsiveness in several mouse models. We have identified the polymorphism responsible for two of these loci. Using composite interval mapping and multiple interval mapping in crosses involving C57BL/6J, A/J, and SJL/J strains, we have confirmed the existence of multiple angiogenesis-response loci on chromosome 7. One of these (AngVq4) localized to the middle of the chromosome and was centered on tyrosinase. Congenic animals confirmed this locus, but could not demonstrate that the classical tyrosinase albino (c) mutation was causative because of the existence of additional linked loci in the congenic. In 1970, a second tyrosinase albino mutation (c-2J) arose in the C57BL/6J background at Jackson Labs. Testing this strain (C57BL/6J) demonstrated that the albino mutation is sufficient to completely explain the alteration in angiogenic response that we observed in congenic animals. Thus, we conclude that the classical tyrosinase mutation is responsible for AngVq4. Similar methods have identified the classical pink-eyed dilution mutation as the genetic change responsible for AngFq5.

Characterizing novel angiogenesis regulating genes detected by QTL mapping may identify additional therapeutic targets for antiangiogenic agents. These experiments also reveal the important effect of the host genetics on angiogenesis and tumor growth. The identification of “high angiogenic” individuals which are at risk for angiogenesis dependent diseases will allow for increased monitoring and better clinical management. Further, new antiangiogenic therapies with low toxicity may allow for the pretreatment of such individuals rendering them “low angiogenic” and thereby increase survival.

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 364.
Abstract 3641: The DNA repair protein hABH2 mediates temozolomide resistance in human glioblastoma cells
Authors: Johannessen Tor-Christian A. , Prestegarden Lars , Tysnes Berit B. , Bjerkvig Rolf
Published: February 2011

Background and Purpose:

Glioblastoma multiforme is the most malignant and frequent primary brain tumour in adults. Current treatment remains insufficient as these tumours display a diffuse infiltrative growth pattern and tend to recur despite extensive debulking surgery followed by radio- and chemotherapy. The methylating agent temozolomide is at present the cornerstone of glioblastoma chemotherapy. However, several independent DNA repair mechanisms can restore the integrity of methylated DNA bases, and thus contribute to temozolomide resistance and subsequent therapy failure. In this work we examined whether human AlkB homologue 2 (hABH2), an oxidative demethylase capable of reversing methyl lesions in DNA, might provoke temozolomide resistance in human glioblastoma cells.

Materials and Methods:

hABH2 expression was examined in eight established glioma cell lines as well as normal human brain using quantitative real-time PCR. Cells derived from a hABH2 deficient glioblastoma were transfected with a hABH2 plasmid prior to antibiotic selection, and subsequent colonies were screened for hABH2 expression by western blotting. Temozolomide sensitivity was assessed for hABH2 proficient and deficient colonies using a tetrazolium salt assay after 96 hours of drug incubation.

Results:

mRNA levels of hABH2 were generally 2-4 fold higher in the established cell lines compared to normal human brain. Furthermore, hABH2 overexpressing glioblastoma cells were significantly more resistant to temozolomide than hABH2 deficient cells derived from the same patient.

Conclusions:

Our results show that hABH2 is expressed at high levels in glioma cells, and that overexpression of this protein mediates temozolomide resistance in vitro.

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 3641.
Abstract 365: Autocrine vascular endothelial growth factor signaling mediates survival and chemoresistance of human colorectal cancer cells
Authors: Samuel Shaija , Fan Fan , Dang Long H. , Gaur Puja , Xia Ling , Zhou Yunfei , Liu Jia , Tozzi Federico , Sceusi Eric , Ye Xiang-Cang , Ellis Lee M.
Published: February 2011

Introduction. Although the effects of VEGF on angiogenesis and vascular function are well known, the autocrine effects of VEGF on tumor cell function remain to be elucidated. We studied phenotypic changes in human colorectal cancer (CRC) cells with homologous deletion of VEGF alleles to determine any potential autocrine/intracrine role of VEGF on tumor cell function.

Methods. The human CRC cell lines HCT116 and LS174T with homologous recombination-mediated deletion of VEGF alleles were generated previously (Dang et al, Can Res 2006). To exclude the effect of exogenous VEGF, the cells were grown in low serum condition for all assays. MTT assay was used to detect changes in cell growth. Migration and invasion were determined using modified Boyden chamber assays. Cell death and apoptosis were assessed by FACS analysis following PI and Annexin V/PI staining respectively. Changes in protein levels of VEGF signaling intermediates and mediators of apoptosis were evaluated by Western blotting. Chemosensitivity was determined by FACS analysis following 5-FU treatment and PI staining of cells.

Results. Loss of VEGF expression decreased cell growth and increased spontaneous apoptosis in CRC cells. Cells null for VEGF demonstrated increases in migration and invasion. Loss of VEGF expression also increased the sensitivity of cells to the cytotoxic effects of the chemotherapeutic drug, 5-FU, as shown by increased cell death and apoptosis. These effects are mediated via upregulation of the pro-apoptotic mediators cleaved caspase-3 and PARP and, downregulation of the anti-apoptotic mediator survivin. In addition, loss of VEGF led to increased levels of PlGF, SDF-1 and phosphorylated VEGFR-1 in CRC cells.

Conclusion. Loss of autocrine/intracrine VEGF signaling leads to an increase in spontaneous apoptosis and chemosensitivity of colon cancer cells. In addition, loss of VEGF signaling led to increases in PlGF and SDF-1, suggesting that loss of VEGF signaling can lead to induction of compensatory pathways that may be mediated by numerous cell types in the tumor microenvironment. These findings have implications for a better understanding of mechanisms of action for VEGF targeted therapies when combined with chemotherapy in patients with metastatic CRC.

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 365.
Abstract 3658: 5F 203 modulates oxidative stress, induces single-strand break formation and promotes mitochondrial membrane potential loss in sensitive breast cancer cells
Authors: Brantley Eileen J. , Watkins Cheri , McLean Lancelot , Amis Louisa , Davis Willie
Published: February 2011

Targeted pharmacotherapeutic agents have been successful in treating women with breast cancer that either over-expresses human epidermal growth factor receptor 2 (Her2/neu) or is dependent upon estrogen receptor signaling. However, patients with Her2/neu-negative or hormone-independent breast cancer do not benefit from these targeted agents. A candidate anticancer agent 2-(4-amino-3-methylphenyl)-5-benzothiazole (5F 203) targets the aryl hydrocarbon receptor signaling pathway and potently inhibits the growth of breast cancer cells irrespective of either estrogen receptor or Her2/neu status. 5F 203 may represent a promising agent for women who lack targeted therapeutic options. We have previously assessed the role reactive oxygen species (ROS) play in mediating the anticancer activity of 5F 203. In our current studies, we pretreated 5F 203-sensitive breast cancer cells with rotenone prior to treatment with 5F 203. This pretreatment diminished 5F 203-mediated ROS formation as measured by DCF-DA staining followed by flow cytometry. This suggests 5F 203-induced ROS production is partially derived from the mitochondria. Using the comet assay and a JC1 dye-based fluorometry assay respectively, we found 5F 203 induced single-strand break formation and mitochondrial membrane potential loss in sensitive breast cancer cells. Pretreatment with the antioxidant N-acetyl-L-cysteine partially diminished apoptosis as assessed by relief contrast microscopy and the Annexin V/PI assay. This implies that 5F 203-induced apoptosis depends at least in part on ROS derived from mitochondria. We also analyzed 84 oxidative stress-responsive genes using PCR-array and determined that 5F 203 significantly increased the expression of cytoglobin, a novel tumor suppressor, in sensitive breast cancer cells. These data imply ROS and cytoglobin contribute to the anticancer activity of 5F 203.

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 3658.
Abstract 3663: Effects of chemotherapy on cell survival, cell cycle biology and apoptosis in the SK-N-FI neuroblastoma cell line
Authors: Blanco Idoia , Lazcoz Paula , Celay Jon , Fan Xing , Castresana Javier S. , Encio Ignacio J.
Published: February 2011

Neuroblastoma is a malignant embryonal childhood tumor of the peripheral nervous system, which often shows poor clinical prognosis. p53 mutations are rare at diagnosis of neuroblastoma but may be acquired at relapse, as well as chemoresistance.

We have previously detected a mutation in exon 5 of the p53 gene (codon 135; Cys to Phe) in the SK-N-FI neuroblastoma cell line. To test whether this mutation affects p53 function driving the cells to a higher chemoresistant phenotype, we analyzed the action of doxorubicin, etoposide, cisplatin, melphalan and retinoic acid on cell survival, apoptosis induction and cell cycle distribution in these cells.

SK-N-FI cells were incubated in the presence of doxorubicin, etoposide, cisplatin, melphalan and 9-cis or all-trans retinoic acid at five different concentrations ranging from 10-4 to 10-8 M. After 72 h of treatment, cell survival was estimated by the MTT method. Quantification of apoptosis and cell cycle analysis were carried out by flow cytometry. With this purpose, fixation of SK-N-FI cells was conducted after 12, 24, 72 h and 5 days of treatment with the previously mentioned drugs.

All tested compounds were toxic at the micromolar range except for the retinoic acid isomers which were active at higher doses. The smaller GI50 values were found with doxorubicin, followed by melphalan, etoposide and cisplatin.

A statistically significant rise in apoptosis was observed in all cases at day 5. This increase was even noticeable at 72 h for doxorubicin, 9-cis retinoic acid and cisplatin.

With regard to the cell cycle distribution, a decrease of the G0/G1 cell population at day 5, accompanied by a time course increase in the Sub-G1 phase, was detected for every tested drug but the retinoic acid isomers. Melphalan also decreased S phase cells. Moreover, while for doxorubicin treatment reduction of the G0/G1 cell population was preceded by an increase in S and G2/M cells at shorter periods, for etoposide and cisplatin only the reduction in the G0/G1 cells in the long term was observed. Finally, 9-cis retinoic acid but not all-trans retinoic acid treatment provoked an initial accumulation of G0/G1 cells that was followed by an increment in the Sub-G1 phase.

Despite bearing a mutation of the p53 gene, SK-N-FI cells respond to doxorubicin, cisplatin, etoposide, melphalan, 9-cis and all-trans retinoic acid. All mentioned drugs were able to induce apoptosis, affected to the cell cycle distribution of the cells in a drug specific manner and, except for the retinoic acid isomers, displayed cytotoxic activity at the micromolar range.

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 3663.
Abstract 3675: Farnesyltransferase inhibitor (FTI) disruption of Rheb/FKBP38 association leads to FKBP38-mediated inactivation of mTOR
Authors: Yang Hua , Wang De-An , Hamilton Andrew D. , Sebti Said M.
Published: February 2011

The activation of the Rheb (Ras homologue enriched in brain) / mTOR (mammalian target of rapamycin) pathway plays a central role in integrating growth factor, nutrient and energy signals and regulates cell growth (cell mass and size) and proliferation (cell number). Recently Rheb was found to activate mTOR by binding and antagonizing its endogenous inhibitor, FKBP38. However, it is not known whether inhibition of Rheb farnesylation by farnesyltransferase Inhibitors (FTIs) is sufficient to disrupt Rheb/FKBP38 interaction and leads to inactivation of mTOR. Here we demonstrate that FTI-2153 disrupts Rheb binding to FKBP38 allowing FKBP38 to bind and to inactivate mTOR. Furthermore, in human cancer cells, FTI-2153 also blocks IGF-1-stimulated Rheb/FKBP38 binding and promotes mTOR/FKBP38 binding. We further show that FTI-2153 inhibits Rheb-mediated activation of mTOR to phosphorylate S6 kinase, and that a geranylgeranylated mutant variant of Rheb that is as efficient as wild-type farnesylated Rheb at activating mTOR rescues cancer cells from FTI-induced inactivation of mTOR signaling, inhibition of anchorage-dependent and -independent tumor cell growth and induction of apoptosis. These results demonstrate that FTI-mediated disruption of the Rheb/FKBP38 interaction leads to inactivation of mTOR and is an effective approach to inhibiting tumor proliferation and survival and that inhibition of Rheb farnesylation mediates, at least in part, the anti-tumor effects of FTIs.

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 3675.
Abstract 3676: A selective small molecule inhibitor of c-Jun N-terminal kinase 1
Authors: Yao Ke , Cho Yong-Yeon , Bode Ann M. , Vammenthala Anuradha , Park Jewn Giew , Liu Kangdong
Published: February 2011

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein (MAP) kinase family. The jnk1 and jnk2 genes are expressed ubiquitously, whereas jnk3 has a limited pattern of expression and is largely restricted to brain, heart, and testis. The JNKs signal transduction pathway was shown to play an important role in coordinating various cellular responses including apoptosis, proliferation, and neoplastic transformation. Indiscriminately suppressing the activity of all three c-Jun N-terminal kinase isoforms, JNK1, JNK2, and JNK3, is probably not an appropriate strategy because each JNK appears to have a distinct function in cancer, asthma, diabetes, and Parkinson's disease. However, until now, isoform selective inhibitors for JNK1 over JNK2 or JNK3 activities have not been developed. Therefore, using structure-based design, we developed (in-house) a selective isoform inhibitor that showed a selective inhibitory effect against JNK1, but not JNK2 or JNK3. We found that 7-(6-N-Phenylaminohexyl)amino-2H-anthra[1,9-cd]pyrazol-6-one (AV-7) suppressed JNK1 activity, with no effect on either JNK2 or JNK3, in vitro. In cell culture systems, we also found that AV-7 inhibited JNK1 activity dose-dependently. In contrast to SP600125, a non-selective JNKs inhibitor, that binds to the ATP active site, AV-7 was designed to target the protein-protein interaction between JNK1 and its binding partner(s) rather than the ATP binding site. At 10 μM, AV-7 specifically inhibited JNK1 by about 50%. The phosphorylation of c-Jun by JNKs induces c-Jun transactivation and transcriptional activity through the formation of a protein complex with c-Fos or ATF1 (i.e., AP-1 complex). c-Jun/AP-1 enhances transcription by binding to the promoter region of many genes. We found that AV-7 inhibits UVB-induced c-Jun phosphorylation and sub-G1 accumulation in JNK2−/− MEFs, which express only JNK1, but had no effect on c-Jun in JNK1−/− MEFs, which only express JNK2. These results demonstrate that AV-7 is an isoform selective JNK1 inhibitor. In humans, JNKs play a key role in obesity-induced type 2 diabetes mellitus and the absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and an enhanced insulin receptor signaling capacity in mouse obesity models. Therefore, we suggest that because of JNK1's critical role in diabetes, AV-7 might have therapeutic potential against this devastating disease.

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 3676.
Abstract 3687: Expression of a Src-family kinase in CML cells induces resistance to imatinib in a kinase-dependent manner
Authors: Pene-Dumitrescu Teodora , Smithgall Thomas E.
Published: February 2011

Chronic Myelogenous Leukemia (CML) is characterized by the presence of Philadelphia Chromosome, which results in the expression of the 210 kDa Bcr-Abl tyrosine kinase. Bcr-Abl constitutively activates signaling pathways important for the proliferation, survival and cytokine independence of myeloid progenitors, including the Src family kinases (SFKs). Imatinib, an inhibitor of Bcr-Abl tyrosine kinase activity, is the frontline therapy for CML. Although imatinib is remarkably effective in the chronic phase of the disease, patients with accelerated or blast crisis CML often develop resistance. Several recent studies performed on clinical specimens from imatinib-resistant patients with wild-type Bcr-Abl found that the Src family kinase members Hck and Lyn are overexpressed or highly active, suggesting that Src kinases may play a role in imatinib resistance. While the role of Lyn has been addressed in some detail, the contribution of Hck to this type of imatinib resistance is less well understood. To test whether Hck overexpression in CML cells induces resistance to imatinib in a kinase-dependent manner, we employed a Hck mutant (Hck-T338A) that is uniquely sensitive to the pyrazolo-pyrimidine inhibitor, NaPP1. In vitro, Hck-T338A was 48 times more sensitive to NaPP1 than the wild-type kinase. Importantly, this “gatekeeper” mutation was functionally silent and did not confer a loss or gain of function on Hck in a control fibroblast transformation assay. Expression of wild-type Hck or the T338A mutant in K562 CML cells resulted in resistance to imatinib-induced apoptosis and inhibition of soft-agar colony formation. Treatment with NaPP1 restored sensitivity to imatinib in a concentration-dependent manner only in cells expressing the Hck-T338A mutant. In contrast, cells expressing wild-type Hck were not affected by NaPP1, demonstrating the selectivity of NaPP1 for the Hck-T338A mutant. This result shows that Hck-induced imatinib resistance requires Hck kinase activity. In addition, NaPP1 also reduced Hck-mediated phosphorylation of Bcr-Abl at sites that may affect imatinib binding exclusively in cells expressing Hck-T338A. Together, these data establish a direct cause and effect relationship between Hck overexpression and imatinib resistance in CML cells. Furthermore, our results show that imatinib resistance requires Hck kinase function and may occur via direct Hck-induced trans-phosphorylation of Bcr-Abl. Selective drug targeting of Hck may be of therapeutic benefit in imatinib-resistant CML patients with increased Hck expression or activity.

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 3687.
Abstract 3688: A flexible kinase inhibitor assay for active, non-activated, and impure kinase preparations
Authors: Riddle Steven , Lebakken Connie , Ellefson Jason , Reichling Laurie , Wolken Jill
Published: February 2011

Kinase activity-based assays are cost-effective and widely used to drive early drug discovery and lead identification, however, they typically require purified, active kinase preparations to be produced that are shown to phosphorylate a known substrate. While it is possible to satisfy these requirements for well-studied kinases, there are many kinases which lack one or more of these requirements, such as CDK8 and STK33, making drug discovery efforts difficult. To solve for these assay development problems, we have developed a TR-FRET binding assay platform which allows for characterization of compounds with kinases that are not sufficiently purified, completely lack activity, or do not have a known substrate. These new assay options for addressing kinase assay requirements in early drug discovery should enable more targets to be screened sooner, resulting in compounds being made available for lead optimization faster. The ability to detect and characterize binding of compounds to non-activated kinases is also a benefit to the lead optimization process, as compounds that bind preferentially to kinases in their non-activated state, such as Gleevec®, are generally preferred due to their selectivity. In this poster, we present solutions for kinases that are difficult to address with activity assays and provide a comparison of compound affinities for active and non-activated forms of a diverse set of kinases to enable faster lead optimization.

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 3688.
Abstract 3693: Small molecule microarray screens for inhibitors of oncogenic ETS factors
Authors: Pop Marius , Doug Katie , Bradner James , Koehler Angela , Garraway Levi
Published: February 2011

Members of the ETS transcription factors family are involved in many cancers and are often targeted by translocation events that result in oncogenic gene fusions or dysregulated ETS factor expression. For example, the vast majority of prostate cancers harbor translocations involving the ETS factors ERG, ETV1 and ETV4 (most commonly, these are fused to the TMPRSS2 androgen-regulated gene or other house keeping genes). Overexpression of ETS factor gene fusions can induce or sustain malignant characteristics of cancer cells such as survival or invasiveness. However transcription factors have often been deemed “undruggable” by conventional approaches.

The focus of this project is to employ novel screening approaches to identify and characterize small molecules that are able to bind and modulate the biological function of oncogenic ETS factors. Toward this end, we have performed Small Molecule Microarray (SMM) screens that involve a total of 80,000 distinct small molecules spanning several categories, including diversity-oriented synthesis (DOS) products, natural products, and other biologically active chemotypes. The SMM screens identified six compound that selectively bind the ETS factor ETV1 with a stringent Zscore. Two of these compounds have shown inhibitory activity in an ETV1-specific promoter-induced luciferase system. Surface Plasmon Resonance analysis has confirmed that each of the two lead compounds binds ETV1 directly, with one compound showing submicromolar binding kinetics. This compound in particular was able to reduce the expression of several ETV1 downstream targets such as MMP1 and MMP7. These efforts suggest that we have identified novel small molecule “perturbagens” of ETV1, an oncogenic ETS transcription factor. More generally, our results suggest that SMM-based screens may offer a robust approach to identify novel compounds that bind and perturb the function of cancer-associated proteins deemed poorly “druggable” by conventional methods.

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 3693.
Abstract 370: Characterization of human umbilical vein endothelial cell clones resistant to VEGFR2 tyrosine kinase inhibitor
Authors: Matsumoto Kazuko , Arao Tokuzo , Furuta Kazuyuki , Sakai Kazuko , Nagai Tomoyuki , Kudo Kanae , Kaneda Hiroyasu , Tamura Daisuke , Aomatsu Keiichi , Fujita Yoshihiko , Nishio Kazuto
Published: February 2011

Acquired resistance to anti-angiogenetic drugs have emerged as a potentially important problem in clinical settings, however, the underlying mechanism remains largely unclear. In this study, we established human umbilical vein endothelial cell (HUVEC) clones that are resistant to VEGFR tyrosine kinase inhibitor (VEGFR-TKI, Ki8751) using MNNG and VEGFR-TKI.

In growth inhibition assay, VEGFR-TKI resistant HUVEC clones were significantly decreased cellular sensitivity to Ki8751 about 4-10 fold compared with that in the parental cells. VEGF-mediated cellular proliferations of resistant clones were also decreased. Interestingly, VEGFR2 expression was down-regulated and the phosphorylation levels of Akt were up-regulated in resistant clones. Finally, microarray analysis revealed that several angiogenesis-related or -specific genes including CD31 were remarkably down-regulated in resistant clones compared with the parental cells. In conclusion, these results suggest that the mechanism of VEGFR-TKI resistant is closely involved in “escape phenomenon” from VEGF-VEGFR system and our findings provide a novel insight into the drug resistant to VEGFR-TKI in vascular endothelial 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 370.