bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4445: Pegylated arginine deiminase induces mitochondrial apoptosis and synergizes with cisplatin in ASS1-negative malignant pleural mesothelioma
Authors: Delage Barbara , Chelala Claude , Chacko Alex , Jithesh Puthen V. , Fennell Dean A. , Lemoine Nicholas R. , Szlosarek Peter W.
Published: February 2011

We have explored the role of pegylated arginine deiminase (ADI-PEG20), an arginine catabolizing enzyme, in argininosuccinate synthetase 1 (ASS1)-negative malignant pleural mesothelioma (MPM). MPM is a chemorefractory disease, increasing worldwide, with a median survival of less than 1 year. Novel treatments are urgently required. MPM tumors, lacking ASS1, are auxotrophic for arginine and therefore sensitive to arginine depletion driven by ADI-PEG20.

First we assessed the impact of ADI-PEG20 on global gene expression to identify novel interacting pathways in the ASS1-negative MPM cell line, JU77, using the human genome U133 Plus 2.0 Array from Affymetrix combined with bioinformatic analysis. Next, three ASS1-negative MPM cell lines (MSTO-211H, 2591 and JU77) were treated in triplicate with different fixed ratios of ADI-PEG20 and cisplatin. Cellular viability was determined 6 days post-treatment by MTS colorimetric assay. Apoptosis protein induction by ADI-PEG20 and platinum, alone and in combination, was measured by western blot after mitochondrial and cytosolic fractionation. Drug interactions were analyzed using the isobologram method of Chou and Talalay.

ADI-PEG20 modulated several thousand genes involved in cell cycle, DNA damage, immune, and inflammatory pathways in the ASS1-negative JU77 MPM cell line by 24hrs of drug treatment. In contrast, stable transfection of ASS1 cDNA in ASS1-negative MPM cell lines resulted in resistance to ADI-PEG20 with minimal evidence of gene modulation by 24hr of drug treatment as assessed in the JU77 MPM cell line. ADI-PEG20 triggered mitochondria-dependent apoptosis as evidenced by Smac release from mitochondria into the cytosol of ASS1-negative MPM cell lines. We confirmed that arginine depletion by ADI-PEG20 altered the mTOR/p70S6K signaling pathway and led to dephosphorylation of the translation regulation proteins S6K and E4-BP1. The expression of cell cycle regulating proteins p21 and cyclin D1 was decreased by arginine depletion, alone and in combination with cisplatin. The enhancement of cisplatin cytotoxicity by ADI-PEG20 with loss of ASS1-negative MPM cell viability as early as 24hr post treatment, supports the demonstrated strong synergy between the two drugs.

In summary, the antitumor effect of platinum was potentiated markedly using ADI-PEG20, suggesting that combining arginine depletion with the current standard of care, platinum and antifolates, may offer an improvement over doublet chemotherapy alone.

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 4445.
Abstract 4448: Disruption of nedd4 and connexin 46 interaction by gap junction activator in breast cancer cells
Authors: Nguyen Thu Annelise , Ding Ying , Bigelow Kristina
Published: February 2011

Breast cancer is the second most common cancer among women; it is also the second leading cause of cancer death among women in the United States. Progress in understanding the etiology of this disease and developing therapies has been slow due to multiple deregulations of various genes. Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). Most normal cells have functional gap junctional intercellular communication (GJIC), while most, if not all, tumors cells have dysfunctional GJIC. GJIC consists of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, cell signaling and previous studies have been suggested their participation in tumor formation and progression. It is believed that restoring GJIC is linked to drug sensitivity and reduction of tumorigenicity. Thus, increasing gap junction activity or enhancing GJIC in tumor cells provides the targets to enhance anti-neoplastic therapies. Several GJIC enhancers have been reported; however, an effective clinical drug targeting gap junction is not available at this time. Recently, we synthesized a new class of substituted quinolines (code name: PQ) and found that they possess potent inhibitory activities against T47D breast cancer cells (IC50 value of PQ1 is 119 nM) through the enhancement of GJIC. Since the mechanism of action of GJIC enhancement caused by PQ is unknown, the current study is to determine the target proteins that PQ interacts with, which may lead to the mechanism of enhancement of GJIC and inhibition of cancer cell growth. Using surface plasmon resonance (SPR) spectroscopy, we have found that PQ1 has a strong binding with Nedd4, E3 ubiquitin ligase which previously been shown to involve in the degradation of gap junction. T47D breast cancer cells were treated with various concentrations of PQ1 for 24 hours. Whole cell extract was isolated and subjected to immunoprecipitation assay, followed by western blot analysis. The results show that 100 nM PQ1 can cause a 2-fold decrease in the interaction of Nedd4 and connexin 46 compared to control without treatment or solvent. Since gap junction turnover has been shown to be facilitated by lysosomal and proteasomal degradation, disruption of Nedd4 and connexin 46 suggests that PQ1 may inhibit the lysosomal and proteasomal degradation of connexin 46 in breast cancer cells. Future study will focus on whether PQ causes an increase in gap junction activity via inhibition of Nedd4-ubiquination of connexin.

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 4448.
Abstract 4449: Effects of endocrine therapy on mRNA- and microRNA expression profiles in prostate cancer
Authors: Lehmusvaara Saara , Erkkilä Timo , Lähdesmäki Harri , Kujala Paula , Tammela Teuvo L.J. , Visakorpi Tapio
Published: February 2011

Introduction & Objectives: The gold standard treatment for advanced prostate cancer is endocrine therapy either by castration (surgical or chemical) or with antiandrogens. Although endocrine therapy has been used for decades, the molecular consequences of androgen deprivation are poorly understood. The aim of this study was to investigate how endocrine therapy affects genome-wide gene expression and to ascertain whether chemical castration or antiandrogen use leads to unique gene expression profiles.

Methods: 30 patients were randomized equally into three groups: no treatment, bicalutamide (antiandrogen) 150 mg daily, and goserelin (GnRH agonist) 3.6 mg every 4 weeks. Following the neoadjuvant treatment for 12 weeks, patients underwent radical prostatectomy. Freshly frozen specimens were collected for expression profiling of both protein (mRNA) and microRNA (miRNAs) coding genes using Illumina (probes for ∼25 000 mRNAs) and Agilent (723 miRNAs) microarrays, respectively. The amount of cancer, benign epithelium, and stroma in the specimens was assessed using H&E stained slides. Statistical analyses were first done without taken into account the tissue composition of specimens. Subsequently, tailor-made in silico Bayesian modeling tool was used to predict the mRNA and miRNA expression patterns in the different cellular compartments. mRNA expression from microarray data and in silico predictions were validated with qPCR from microdissected tissue samples.

Results: In unsupervised hierarchical clustering of mRNA profiles, specimens from bicalutamide and goserelin arms were clustered separately from control samples, but not separately from each other. Out of 600 significantly differently expressed mRNAs between control and treatment arms (p<0.05), only 56 were unique for bicalutamide treatment and 13 for goserelin treatment. Interestingly, tissue compartment analysis showed that bicalutamide affects more specifically cancer cells while goserelin has more effect to stroma and normal epithelium. In the miRNA profiles, there were only minor changes in the bicalutamide and goserelin treatment arms compared with control arm. Only 6 miRNAs were significantly differently expressed (p<0.05). Also, there was more variety in miRNA expression between different tissue compartments than treatment arms.

Conclusions: In conclusion, androgen deprivation had more effect on mRNA than miRNA expression. Most of the differentially expressed genes behaved similarly after GnRH agonist and antiandrogen treatments.

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 4449.
Abstract 4450: A new paradigm for AS1411 activity: Uptake by macropinocytosis and induction of macropinocytosis by a nucleolin-dependent mechanism
Authors: Reyes-Reyes Elsa M. , Bates Paula J.
Published: February 2011

AS1411 is an experimental drug that is currently in Phase II clinical trials as a novel therapy for cancer. It is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. We have shown previously that AS1411 can efficiently inhibit proliferation and induce cell death in many types of cancer cells, but has little effect on normal cells. Here, we report on the uptake of fluorescently labeled AS1411 (FL-AS1411) in a variety of cancer cell lines (sensitive to AS1411) and non-malignant cells (resistant to AS1411), which we studied using flow cytometry and confocal microscopy. In both cancer and non-malignant cells, uptake of FL-AS1411 was much higher that that of an inactive control oligonucleotide and was rapid (clearly visible in cytoplasmic foci by 15 min). Uptake of FL-AS1411 (evaluated at 2 h) was independent of the presence of serum, but strongly temperature-dependent, indicating an active process. In cancer cells, FL-AS1411 uptake was significantly suppressed by macropinocytosis inhibitor, amiloride, but not by dynamin inhibitor, dynasore, and confocal microscopy showed that AS1411 co-localized mainly with the macropinocytic tracker, dextran, but not with transferrin (a marker of receptor-mediated endocytosis). In contrast, in non-malignant cells, FL-AS1411 uptake was significantly suppressed by dynasore, but not by amiloride. These results suggest that the major mechanism of internalization of AS1411 occurs via macropinocytosis in cancer cells, but via clathrin-dependent and/or caveolae-dependent pathways in normal cells. Additionally, we found that treatment of cancer cells with an oligonucleotide containing the AS1411 sequence caused hyperstimulation of macropinocytosis, provoking an increment in its own uptake. No stimulation of macropinocytosis was observed in non-malignant cells, which are insensitive to AS1411. By using siRNA approaches, we showed that nucleolin is not required for the initial phase of FL-AS1411 uptake (at 2 h), but it is necessary for the induced macropinocytosis and consequently for the second phase of FL-AS1411 uptake (at 24 h). These data confirm the important role of nucleolin in mediating the activity of AS1411 and provide a new model for the mechanism of action of AS1411 in cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4450.
Abstract 4458: Antiproliferative effects of γ-tocotrienol are mediated by suppression in the activation of Met tyrosine kinase receptors in neoplastic +SA mammary epithelial cells
Authors: Bachawal Sunitha V. , Wali Vikram B. , Sylvester Paul W.
Published: February 2011

γ-Tocotrienol, a member of vitamin E family of compounds, displays potent anticancer activity against breast and other types of cancers. Previous studies have shown that the antiproliferative effects of γ-tocotrienol are associated with the suppression in the epidermal growth factor (EGF)-dependent phosphatidylinositol-3-kinase (PI3K)-dependent kinase-1 (PDK-1) and Akt phosphorylation in neoplastic +SA mammary epithelial cells. Also, studies showed that γ-tocotrienol treatment had no direct effect on Akt activity and did not alter the relative intracellular levels of PI3K, indicating that γ-tocotrienol might act upstream of the PI3K/PDK-1/Akt pathways at the level of membrane tyrosine kinase receptors in +SA mammary tumor cells. Since aberrant activation of the Met tyrosine kinase receptors is observed in various types of cancers and is associated with poor prognosis, studies were conducted to determine the effect of γ-tocotrienol on EGF and hepatocyte growth factor (HGF)-dependent activation of Met tyrosine kinase receptors in +SA mammary tumor cells. Treatment with serum-free media containing various doses of EGF (1-10 ng/ml) or HGF (1-10 ng/ml) significantly increased the growth of +SA mammary tumor cells as compared to serum-free control group over a 3-day culture period. Stimulation of +SA cells with EGF (10 ng/ml) or HGF (10 ng/ml) resulted in the activation of Met tyrosine kinase receptors. Treatment with Met tyrosine kinase inhibitor, SU11274 (5.5 μM) or γ-tocotrienol (4 μM) inhibited the EGF or HGF-dependent growth and activation of Met receptors in +SA mammary tumor cells after a 3-day culture period. Also, knockdown of Met receptors using Met specific siRNA decreased the expression of Met receptors and significantly inhibited the EGF or HGF-dependent growth of +SA cells. Additional studies showed that when +SA cells were simultaneously exposed to EGF (10 ng/ml) and HGF (10 ng/ml), the growth inhibitory effects of γ-tocotrienol (4 μM) and SU11274 (5.5 μM) were significantly reversed, associated with the activation of Met receptors. However, combined treatment with low doses of γ-tocotrienol (2 μM) and SU11274 (3 μM) inhibited the EGF and/or HGF-dependent growth of neoplastic +SA mammary epithelial cells. These results strongly suggest that γ-tocotrienol treatment may provide significant health benefit in the prevention and/or treatment of breast cancer in women with deregulated HGF/Met signaling. Supported by grants from NIH (CA86833) and First Tech International Ltd.

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 4458.
Abstract 4461: Involvement of cleaved p73 isoforms in anoikis
Authors: Alsafadi Samar , Tourpin Sophie , Benihoud Karim , André Fabrice , Vassal Gilles , Ahomadegbe Jean Charles
Published: February 2011

p73, a p53 family member, is a transcription factor that plays an important role in cell cycle control, differentiation and apoptosis. p73 has been shown to be capable of replacing p53, inducing apoptosis in p53-deficient cells. In this study, we tried to determine the role of p73 in anoikis, a type of apoptosis induced by inadequate cell-matrix interaction. Six breast cancer cell lines with different p53 status were treated with doxorubicin (DOX) and anoikis was analyzed 48 hours after treatment. We found that, independently of the p53 status, DOX-induced anoikis was associated with p73 cleavage and the activation of caspases. p73 cleavage was not triggered in caspase-3-null MCF-7 breast cancer cell line. To confirm the implication of p73 cleavage in anoikis, we transfected breast cancer cell lines with plasmids bearing TAp73α, TAp73β or ΔNp73α. The overexpression of p73 isoforms in transfected cells induced detachment of these cells from the culture flask. Anoikis in these cells was associated with cleavage of the transfected isoform of p73. Once again, p73 cleavage was not detected in caspase-3-null MCF-7 cell line confirming the involvement of caspase 3 in p73 cleavage. The use of flag antibody, which recognizes the N-terminus of transfected proteins, and p73 antibodies, which recognize the C-terminus of these proteins, enabled us to demonstrate that there are two possible cleavage sites in TAp73α generating two different cleaved forms. However, when cells were transfected with TAp73β or ΔNp73α, only one cleaved form was generated. TAp73β probably harbors a N-terminal site, while ΔNp73α only harbors a C-terminal site. Cleavage at the N-terminal site, rather than that at the C-terminal site, appears to be important for an efficient apoptotic response, because it is associated with stabilization of cleaved proteins and high toxicity.

Further studies are warranted to determine the accurate role of p73 cleavage in anoikis, especially since these cleavages arise independently of the p53 status and therefore could contribute to response to stress in cells with non functional p53.

Acknowledgements: this work is supported by “La Ligue contre le cancer - Comité de la Somme” and the Syrian Ministry of Health.

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 4461.
Abstract 4464: C-erbB3 protein modifications are secondary to severe cell membrane alterations induced by Irvalec treatment in CHO cells
Authors: Váradi Tímea , Guijarro José Manuel Molina , Vereb György , Galmarini Carlos M. , Szöllõsi János , Nagy Peter
Published: February 2011

Irvalec® is a marine-derived antitumor agent that is currently undergoing phase II clinical trials. The compound induces a necrotic cell death associated with the appearance of membrane blebs and severe cell swelling reflecting a serious membrane damage caused by the drug. Indirect evidence has also suggested a possible role of ErbB proteins, in particular ErbB3, and lipid rafts in conferring sensitivity to Irvalec treatment. In order to assess the role of ErbB protein expression in the mechanism of action of Irvalec, and to measure the effect of drug treatment on the clustering properties of these proteins, we generated CHO cell lines stably transfected with ErbB2 (CHO-ErbB2) or with ErbB2 and ErbB3 (CHO-ErbB2-3). The IC50 values of the three cell lines for Irvalec did not differ significantly from each other. On the other hand treatment with Irvalec induced a significant increase in the binding of a conformational sensitive antibody to ErbB3 (ErbB3.105.5) without modifying the binding of other ErbB3 antibodies or antibodies against ErbB2. In addition, Irvalec treatment decreased the homoassociation of ErbB2 and ErbB3 measured by flow cytometric FRET. A431 cells transfected with GPI-anchored GFP or ErbB3-citrine showed significant Irvalec-induced patching of the GPI-anchor and ErbB3, whereas the distribution of ErbB1 and ErbB2 were not affected in cells transfected with ErbB1-GFP or ErbB2-mYFP. In conclusion, our data do not support a role for ErbB proteins in determining sensitivity to Irvalec, but show that these receptors are indirectly affected by exposing cells to the compound. These alterations should be ascribed to cell membrane modifications induced by Irvalec treatment

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4464.
Abstract 447: Hypoxia upregulates Nodal expression via the HIF-1 pathway in poorly metastatic breast cancer cells
Authors: Taylor Meghan J. , Postovit Lynne-Marie
Published: February 2011

Low tumour oxygen levels (hypoxia) are associated with a poor clinical prognosis for breast cancer patients. The acquisition of a plastic, stem cell-like phenotype is similarly associated with resistance to therapy and increased metastatic potential. Recent studies have demonstrated that metastatic breast cancer cells express the embryonic stem cell factor Nodal, and that the expression of this protein is correlated with breast cancer progression. Our research establishes that hypoxia induces Nodal expression in poorly metastatic breast cancer cells. The effects of hypoxia on Nodal expression in poorly metastatic MCF-7 and T47D breast cancer cells, which do not endogenously express Nodal, were examined by exposing cells to decreasing concentrations of oxygen (20%-0.5%) for varying amounts of time (0 h - 48 h). Additionally, reoxygenation experiments were performed by exposing cells to 0.5% oxygen for 24 h and restoring them to 20% oxygen for 0 h - 6 days. Time course and dose response experiments determined that T47D cells exposed to 0.5% oxygen for 48 hours saw a maximal (1.5 fold) induction of Nodal mRNA expression and marked increase in protein levels. This expression persisted for up to 6 days with reoxygenation at 20% oxygen. The molecular mechanism underlying the hypoxic induction of Nodal expression was examined by investigating the role of the HIF-1 pathway. HIF-1α expression was knocked down with siRNA and cells exposed to 0.5% oxygen, and conversely, HIF-1α was over-expressed and cells exposed to 20% oxygen. The HIF-1α-specific siRNA returned Nodal expression to control levels in hypoxia and over-expression of HIF-1α elicited an upregulation of Nodal expression in normoxic conditions. This work reveals a mechanism by which a normally stem cell-associated factor can be upregulated by hypoxia in poorly metastatic breast cancer cells. Furthermore, it provides additional insight into the oxygen-mediated regulation of cancer cell plasticity and metastatic potential.

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 447.
Abstract 448: Elevated co-expression of HIF-1α and GLUT-1 is associated with poor prognosis for oral squamous cell carcinoma patients
Authors: Eckert Alexander W. , Lautner Matthias HW , Schütze Andreas , Schubert Johannes , Bilkenroth Udo , Taubert Helge
Published: February 2011

HIF1α is the key transcriptional regulator of the response to hypoxia in tumors. GLUT-1 is a HIF1α-dependent target gene that is upregulated under hypoxic conditions and enables glucose transport into tumor cells. We sought to identify if co-expression of both of these hypoxia-related proteins, as detected by immunohistochemistry, had prognostic relevance in oral squamous cell carcinomas [OSCC]. Eighty-two OSCC tumor samples were analyzed for their expression levels of both HIF1α and GLUT-1 by immunohistochemistry. Protein expression was assessed using an immunoreactive score system (IRS) and the correlation between gene expression and both clinical and pathohistological parameters were examined. Overexpression of either GLUT-1 or HIF1α was associated with poor overall survival in OSCC patients. Multivariate Cox's proportional-hazards regression analysis (adjusted for tumor size and tumor grade) revealed that moderate expression of GLUT-1 or HIF1α was significantly associated with overall survival (RR=5.07, p=0.002 and RR=4.5, p=0.017, respectively) as compared to the groups with low levels of expression of GLUT-1 or HIF1α. Co-expression of both HIF1α and GLUT-1 were additively and significantly associated with adverse prognoses in patients with OSCC. Patients whose tumors had high levels of expression of both HIF1α and GLUT-1 were found to have a 10.2-fold increased risk of tumor-related death (p=0.001) in the multivariate Cox's proportional-hazards regression analysis. Co-expression of high levels of HIF1α and GLUT-1 is significantly correlated with prognosis in OSCC patients, suggesting that the co-expression of these proteins can be used as both an early diagnostic and independent prognostic marker.

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 448.
Abstract 4480: Preclinical evaluation of the mTOR inhibitor, temsirolimus, in combination with the epothilone B analog, ixabepilone
Authors: Menefee Michael E. , Bible Keith C. , Roemeling Christina Von , Copland John Al
Published: February 2011

Introduction: Despite significant progress over the last decade in the development of therapeutics for metastatic cancers including breast cancer (MBC), non small cell lung cancer (NSCLC), and renal cell carcinoma (RCC), most patients will ultimately succumb to their malignancy; more effective therapeutics are sorely needed. The microtubule (MT) is a well-validated target for anti-cancer therapeutics. Ixabepilone (Ixa), a MT stabilizer is approved for MBC and has demonstrated efficacy against other solid tumors. mTOR is another validated target for anti-cancer therapeutics. Temsirolimus (Tem) inhibits mTOR and is approved for advanced RCC. We tested the hypothesis that the addition of Ixa to Tem might augment its anti-tumor activity in vitro. Methods: The cell lines, A498 (RCC), MDA-MB-231 (MBC), and A549 (NSCLC) were treated with Tem alone, Ixa alone, or the combination. All cell lines were also treated with the combination of Tem and Ixa in a fixed ratio of 1:20; Tem doses ranged from 0.125 [nM] to 5 [nM] and Ixa doses ranged from 2.5 [nM] to 100 [nM]. The effects of the treatments were evaluated with clonogenic assays. Synergism was evaluated using CalucSyn 2.0 in accord with the isobologram method of Chou and Talalay. Results: Both Tem and Ixa inhibited cell growth in all tested cell lines as single agents in dose-dependent fashions. Moreover, the combination of Tem and Ixa synergistically enhanced cytotoxicity in all tested lines (see table below).Cell LineED50 [nM]CI (lowest value)A4980.449280.404A5490.285440.263MDA-MB-2310.483550.373

Conclusion: The combination of Ixa and Tem displayed promising activity in all tested cell lines. These findings support the further evaluation of this combination in a phase I clinical trial.

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 4480.
Abstract 4482: Novel and selective small molecule inhibitors of 3-phosphoinositide-dependent kinase-1 inhibit the PDK-1/AKT signaling pathway and cell proliferation
Authors: Baxi Sangita M. , Engstrom Lars , Walls Marlena , Marx Matthew A. , Murphy Sean , Ermolieff Jacques , Lingardo Laura , Lam Hieu , Zou Aihua , Alton Gordon , Smeal Tod , Yin Min-Jean
Published: February 2011

Receptor tyrosine kinases (RTKs), PTEN (phosphatase and tensin homolog deleted on chromosome 10), and PIK3CA (encodes the p110α subunit of phosphatidylinositol 3-kinase [PI3K]) frequently contribute to tumor progression through their ability to regulate the intracellular level of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-phosphoinositide-dependent kinase-1 (PDK-1), a serine/threonine kinase, activates the catalytic domain of numerous kinases by phosphorylating their T-loop sites. PDK-1 activity is required for activation of AKT, p70S6K, and RSK which lead to cell proliferation and transformation. The interaction of the pleckstrin homology (PH) domain of AKT with the membrane bound PIP3 confers a conformational change in AKT, allowing PDK-1 to phosphorylate AKT at the residue threonine-308 (T308). This T-loop activation at T308, along with the phosphorylation of the serine-473 residue by mTOR, fully activates the AKT pathway. Although the roles of many PDK-1 substrates have yet to be characterized, the oncogenic activity of aberrant PI3K pathway signaling through PDK-1 has been extensively validated. We have developed a series of 3-Carbonyl-4-Amino-Pyrrolopyrimidne (CAP) compounds that are potent inhibitors of human PDK-1 (full length and kinase domain) which demonstrate more than 100-fold selectivity against P70S6K, PI3K, AKT, and mTOR. In this study, representative compounds from the CAP series were used to perform a variety of anti-tumor assays. We demonstrate PDK-1 compounds inhibit the phosphorylation of T308 on AKT as well as downstream molecules of the PI3K pathway, such as S6 ribosomal protein (S6RP) in breast, lung and colon cancer cell lines harboring a PI3KCA mutation. Additionally, blockade of AKT and molecules in the PI3K pathway leads to the inhibition of cell proliferation and cell transformation in cancer cells. Our data suggest that the inhibition of PDK-1 activity is sufficient to induce anti-tumor activity in cancer cells through the PI3K-PDK1-AKT axis, and that a potent and specific PDK-1 inhibitor could potentially be developed as a therapeutic agent against several cancer types.

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 4482.
Abstract 4487: OSI-027, a potent and selective small molecule mTORC1/mTORC2 kinase inhibitor is mechanistically distinct from rapamycin
Authors: Bhagwat Shripad V. , Crew Andrew P. , Gokhale Praful C. , Yao Yan , Kahler Jennifer , Epstein David M. , Wild Robert , Pachter Jonathan A.
Published: February 2011

The PI3K/AKT/mTOR pathway is frequently activated in many human cancers and mTOR has been clinically validated as an effective anti-cancer target by rapamycin analogs such as CCI-779, RAD001 and AP23573. We discovered OSI-027, a potent and selective small molecule mTOR kinase inhibitor that inhibits both mTORC1 and mTORC2 unlike rapamycin and its analogs that inhibit only mTORC1. We profiled several human cancer cell lines to compare the mechanistic and functional effects of rapamycin and OSI-027. Rapamycin completely inhibited phosphorylation of S6 (surrogate for S6K1) and only partially inhibited phosphorylation of 4E-BP1 while OSI-027 completely inhibited phosphorylation of both S6 and 4E-BP1. Furthermore, rapamycin only decreased phosphorylation of AKT (S473) in 13% of cell lines and increased phosphorylation of AKT (S473) in 62% of cell lines after 24h treatment. In contrast, OSI-027 significantly reduced phospho-AKT (S473) in all cell lines tested. OSI-027 showed broad-spectrum anti-proliferative activity against both rapamycin-sensitive and -insensitive cell lines. Rapamycin only induced apoptosis in a small subset of tumor cell lines whereas OSI-027 induced apoptosis in the majority of tumor cell lines tested. These in vitro advantages of OSI-027 relative to rapamycin translated to greater tumor growth inhibition in xenograft models in vivo. These results demonstrate that OSI-027, a dual mTORC1/mTORC2 kinase inhibitor provides superior activity across a broad range of preclinical cancer models compared to rapamycin. Accordingly, OSI-027 has been advanced into Phase I clinical trials.

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 4487.
Abstract 4497: NVP-BKM120, a novel inhibitor of phosphoinosotide 3-kinase in Phase I/II clinical trials, shows significant antitumor activity in xenograft and primary tumor models
Authors: Maira Michel , Menezes Daniel , Pecchi Sabina , Shoemaker Kevin , Burger Matthew , Schnell christian , Fritsch christine , Brachmann Saskia , Nagel Tobi , Sellers William R. , Garcia-Echeverria Carlos , Wiesmann Marion , Voliva charles F.
Published: February 2011

A substantial number of epidemiological and experimental studies support an important role for PI3K in the biology of human cancer. The activation of PI3K, and its downstream effectors, has been clearly validated as an essential step for the initiation and maintenance of the tumorigenic phenotype. Parallel to the clinical development of our dual pan-PI3K/mTOR modulators (e.g., NVP-BEZ235), we continued our drug discovery activities to identify PI3K inhibitors with distinct biological and pharmacological profiles. Following a structure-based design strategy, we have identified a new clinical candidate, NVP-BKM120. This 2,6-dimorpholino pyrimidine derivative is a potent pan-PI3K (e.g., IC50= 35 nM, p110α) that does not significantly inhibit other protein or lipid kinases (e.g., IC50= 4.6 μM, mTOR). The compound exhibits potent antiproliferative activity against a broad panel of tumour cell lines by specifically blocking the biological function of PI3K signaling components (e.g. IC50 = 93 nM S473P-Akt in Rat1-p110α cells). The activity of this pan-PI3K inhibitor in cellular settings translates well in in vivo models of human cancer. NVP-BKM120 shows good oral bioavailability in preclinical species and demonstrated significant antitumor activity (growth inhibition or regression) at tolerated doses in xenografts of diverse cancer lineage in mice and rat models. Consistent with its mechanism of action, transient increases in plasma insulin and glucose levels are observed in these studies. Analyses of tumor tissues after acute dosing or at the end of efficacy studies showed a good correlation between compound exposure, PI3K pathway blockade (reduction in P-Akt levels) and antitumor activity. Dose dependent tumor growth delay or regression is also observed in primary tumor models, and, as demonstrated previously with our dual pan-PI3K/mTOR inhibitor NVP-BEZ235, in vivo synergistic activity is observed when NVP-BKM120 is combined with a MEK inhibitor in K-Ras human cancer models. NVP-BKM120 is currently undergoing Phase I/II human clinical trials for the treatment of solid tumors and hematological malignancies.

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 4497.
Abstract 4498: Biological characterization of NVP-BKM120, a novel inhibitor of phosphoinosotide 3-kinase in Phase I/II clinical trials
Authors: Voliva charles F. , Pecchi Sabina , Burger Matthew , Nagel Tobi , Schnell Christian , Fritsch christine , Brachmann saskia , Menezes Daniel , Knapp Mark , Shoemaker Kevin , Wiesmann Marion , Huh kay , Zaror Isabel , Dorsch Marion , Sellers William R. , Garcia-Echeverria Carlos , Maira Michel
Published: February 2011

The PI3K/Akt/mTor signaling pathway plays an important role in controlling cell growth, proliferation and survival. Through various mechanisms, the pathway is frequently dysregulated in human cancers, suggesting the use of PI3K inhibitors as novel targeted anticancer therapeutic agents. To this end, substantial drug discovery efforts have been devoted both in pharmaceutical companies and in academia to identify and develop therapeutic agents able to specifically down regulate PI3K or other components of this pathway in tumors cells. Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional PI3K inhibitors from different chemical classes with more stringent selectivity profiles. The key to achieve these objectives was to pursue a structure-based design approach coupled with intensive pharmacological evaluation of selected compounds during the medicinal chemistry optimization process. Here we report on the biological characterization of the pan-PI3K pyrimidine-derived inhibitor NVP-BKM120. This compound inhibits all four Class I PI3K isoforms (IC50 values in the 35 to 248 nM range) with at least 50-fold selectivity (compared to p110α) towards protein kinases. The compound is also active against the most common somatic PI3Kα mutations (H1047R, E542K and E545K). NVP-BKM120 does not significantly inhibit the related Class III (Vps34) and Class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic and relevant tumor cell lines (e.g., IC50 for S473P-Akt in Rat1-p110α cells of 93 nM). This biological activity correlates with inhibition of various Akt downstream signaling pathway components, and with its anti-proliferative activity. Thus, the compound demonstrates significant, concentration dependent cell growth inhibition and induction of apoptosis in a variety of tumor cancer cells, particularly for those harboring p110α mutants and/or over-expressing erbB2. In addition, NVP-BKM120 demonstrates significant, dose dependent in vivo pharmacodynamic activity as measured by inhibition of p-Akt in relevant xenograft models. The pharmacological, biological and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is currently undergoing Phase 1/II clinical trials in cancer patients.

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 4498.
Abstract 4503: Targeting sphingosine kinase 1 is promising novel strategy against breast cancer
Authors: Nagahashi Masayuki , Ramachandran Subramaniam , Rashid Omar , Milstien Sheldon , Spiegel Sarah , Takabe Kazuaki
Published: February 2011

INTRODUCTION: Sphingosine kinase 1 (SphK1) is one of the key enzymes that regulates levels of the potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P). S1P promotes cancer cell survival, proliferation and angiogenesis. Expression of SphK1 is up-regulated in breast cancer and is associated with cancer progression. The aim of this study is to explore the therapeutic implications of targeting SphK1 for breast cancer treatment. We have examined the role of SphK1 in breast cancer proliferation with an isozyme-specific pharmacological inhibitor of SphK1, SK1-I, which does not inhibit other sphingosine kinases, or by gene silencing of SphK1.

METHODS: 4T1-luc2 cells, murine ER negative breast cancer cells that overexpress luciferase were used. siRNA targeted to a specific sequence of mouse SphK1 mRNA (siSphK1) was used for down-regulation of the gene. Western blotting, qPCR, luciferase and colony formation assays were used to investigate SphK1 expression and responsiveness to SK1-I.

RESULTS: qPCR demonstrated that 4T1-luc2 cells overexpress mRNA of SphK1, SphK2, S1P receptors 1, 2, and 4. Down-regulation of SphK1 mRNA using siSphK1 in these cells was confirmed both by qPCR and Western blotting. siSphK1 suppressed the growth of 4T1 cells determined by luciferase assays which also revealed that SK1-I inhibited 4T1 cell proliferation in a dose-dependent manner. Of note, SK1-I inhibited 4T1 cell growth synergistically with the chemotherapeutic drug doxorubicin. Moreover, siSphK1 or SK1-I also reduced colony formation of these cells.

CONCLUSIONS: Our results support the notion that SphK1 is an important factor in breast cancer proliferation and suggest that SK1-I, an isozyme-specific inhibitor of SphK1, deserves consideration as a new therapeutic agent for this disease. SK1-I could be an important adjunct to anticancer therapy, allowing the use of reduced doses of a cytotoxic chemotherapeutic drug with the same antitumoral efficacy.

This work was supported by NIH grant 5K12HD055881 and Susan G. Komen for the Cure Career Catalyst Research Grant KG090510 to KT, NCI grant R01CA61774 to SS, and Sumitomo Life Social Welfare Services Foundation to MN.

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 4503.
Abstract 4504: Genetic determinants of AKT-dependence in epithelial ovarian cancer
Authors: Hanrahan Aphrothiti J. , Scarperi Stefano , Sakr Rita A. , Westfal Maggie L. , Godsell Jason , Janakiraman Manickam , Levine Douglas A. , She Qing-Bai , Rosen Neal , Solit David B.
Published: February 2011

Ovarian cancer is a genetically complex disease. Recent data compiled by The Cancer Genome Atlas Project highlights the heterogeneity within this disease, though distinct genetic signatures, such as PI3K pathway aberration, KRas alteration and Rb loss, are emerging that may condition treatment response. The high frequency of AKT activation in ovarian cancer, via PTEN mutation/deletion, PIK3CA/AKT mutation/amplification, RAS mutation, or mutation/amplification of upstream receptor tyrosine kinases (RTK), suggests that inhibitors of this pathway may be particularly effective treatments for this disease. We utilized a selective, allosteric inhibitor of AKT to determine the AKT-dependence of a panel of ovarian cancer cell lines. This AKT1/2 inhibitor (Merck AKTi-1/2) has been extensively tested against panels of kinases and PH-domain containing proteins and based upon these studies appears to be highly selective for AKT1 and AKT2. The drug effectively inhibited AKT activity as measured by a decrease in AKT phosphorylation at serine 473 in all cell lines. Of the 17 cell lines tested to date, 3 were hypersensitive (IC50 = 0.1um) to AKT inhibition, while 1 demonstrated intermediate sensitivity (IC50 = 2uM) and 13 were resistant (IC50 >=5uM). Levels of AKT phosphorylation at serine 473 were inversely correlated with Merck AKTi-1/2 IC50 (p = 0.036) but AKT activation alone was insufficient to confer sensitivity. Activation of the MAPK pathway, via KRas/BRaf/MEK1 mutation or KRas amplification, and Rb loss were predictive of resistance to AKT inhibition. In sensitive cell lines, AKT inhibition resulted in hypophosphorylation of RB and accumulation of cells in G1. Significant induction of apoptosis was not observed even in PTEN null, AKT2 amplified and PIK3CA mutant models. Treatment of a PTEN-mutant xenograft model of ovarian cancer with the AKTi-1/2 was also effective in inhibiting AKT phosphorylation and downstream signaling and resulted in a cytostatic tumor response. Ongoing studies are focused on the identification of rational combination strategies that result in induction of apoptosis in the cell lines in which the AKTi-1/2 alone induces growth arrest. The long-term goal will be to identify genetic predictors of AKT-dependence and AKT-inhibitor resistance to guide patient selection for future clinical trials.

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 4504.
Abstract 4505: Akt1 is a potent molecular target for treatment of oral squamous cell carcinoma
Authors: Goda Hiroyuki , Nakashiro Koh-ichi , Hamakawa Hiroyuki
Published: February 2011

Oral cancer has a severe impact on the quality of life (QOL) of patients. Treatment for the advanced cases is given priority for reducing deaths due to oral cancer and keeping QOL after the treatment. Several reports have demonstrated that inactivation of a single critical oncogene in cancer cells can induce the terminal differentiation or apoptosis. In this study, we have attempted to identify a critical oncogene for oral cancer progression. Using Applied Biosystems Human Genome Survey Arrays, we determined the gene expression profiles in 10 oral squamous cell carcinoma (OSCC) primary tissues and 10 human OSCC cell lines. This array contains 32,878 60-mer oligonucleotides probes representing a set of 29,098 individual human genes. We identified Akt1 mRNA that was commonly expressed in all OSCC tissues and cultured cells but not in non-neoplastic tissues and cells. Subsequently, immunohistochemical examination and Western blot analysis showed the overexpression of Akt1 protein in 59 of 63 OSCC primary tissues (94%) but not in oral normal epithelium. Overexpression of Akt1 and phosphorylated Akt1 proteins was also detected in all human OSCC cultured cells by sandwich ELISA. Akt is frequently activated in many types of human cancer, and has been implicated in tumorigenesis and malignancy. To clarify the oncogenic function of Akt1 in human OSCC cells, we used RNA interfering (RNAi) method. We designed and synthesized 8 small interfering RNAs for specific Akt1 (siAkt1), and then tested their RNAi effects. Akt in mammals comprises three highly homologous members known as Akt1, Akt2 and Akt3. Transfection with siAkt1 at the concentration of 1 nM markedly suppressed the expression of Akt1 protein but not Akt2 and Akt3, and significantly reduced the growth rate of human OSCC cells on the plastic surface and in collagen gels. Furthermore, atelocollagen-mediated systemic siAkt1 administration markedly inhibited the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice without interferon responses. These results suggest that Akt1 functions as a critical oncogene in human OSCC cells and targeting Akt1 appears a novel approach for treatment with oral cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4505.
Abstract 4509: Epigallocatechin gallate suppresses tumorigenesis of lung cancer cells by targeting Ras-GTPase-activating protein SH3-domain binding protein 1
Authors: Shim Jung-Hyun , Su Zheng-Yuan , Chae Jung-II , Kim Dong-Joon , Zhu Feng , Ma Wei-Ya , Bode Ann M. , Yang Chung S. , Cho Nam-Pyo , Dong Zigang
Published: February 2011

(−)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified Ras-GTPase-activating protein SH3-domain binding protein 1 (G3BP1) as an important and novel molecular target of EGCG in lung cancer. EGCG interacted with the G3BP1 with high binding affinity (Kd = 0.4 μmol/liter). We also showed that EGCG suppressed anchorage-independent growth of H1299 and CL13 lung cancer cells, which contain an abundance of the G3BP1 protein. EGCG was much less effective in suppressing anchorage-independent growth of H460 lung cancer cells in which G3BP1 levels are low. Knockdown shG3BP1-transfected H1299 cells exhibited substantially decreased proliferation and anchorage-independent growth. shG3BP1-H1299 cells were resistant to the inhibitory effect of EGCG on growth and colony formation compared to shMock-transfected H1299 cells. EGCG interfered with the interaction between G3BP1 and Ras-GTPase-activating protein (Ras-GAP) and further suppressed the activation of Ras. Additional results revealed that EGCG effectively attenuated G3BP1-downstream signaling, including extracellular signaling-regulated kinases, and MAPK kinase in wildtype H1299 and shMock-H1299 cells, but had little effect on H460 or shG3BP1- H1299 cells. Overall, these results strongly indicate that EGCG suppresses lung tumorigenesis through its binding with G3BP1.

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 4509.