bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 281: Novel nucleic acid constructs and formulations for treatment of bladder cancer and malignant ascites
Authors: Liu Yan , Chen Yan , Fosnaugh Kathy , Seth Shaguna , Matsui Yoshiyuki , Harvie Pierrot , Johns Rachel , Takagi Akihide , Vaidyanathan Shobana , Severson Greg , Brown Tod , Zhu Tianying , Charmley Pat , So Alan , Houston Michael , Templin Mike
Published: August 2012

Nucleic acid-based therapeutics offer significant promise in treatment of difficult diseases by providing high sequence specificity against target genes including “non-druggable targets”. Different nucleic acid constructs, single-stranded antisense oligonucleotides, microRNA mimetics, miRNA-specific antagomirs (AntimiR), and double-stranded short-interfering RNAs (siRNAs), provide diverse therapeutic platforms to access targets in treatment of cancer and other diseases. Single-stranded oligonucleotides require additional modifications for increased serum stability, nuclease resistance, and to minimize immune response. Conformationally restricted nucleotide (CRN)-substituted AntimiRs targeting miR-21 and miR-122 demonstrated significant improvement in hybridization affinity with a concomitant increase in potency. A CRN-substituted AntimiR targeting microRNA-122 dosed at up to 50 mg/kg/day over three consecutive days led to a dose-dependent effect on de-repression of downstream liver-specific target genes, Aldolase A (AldoA), Glycogen Synthase I (GYS1) and Solute Carrier Family 7 member 1 (SLC7A1). This CRN-modified AntimiR produced up to a 5-fold increase in AldoA mRNA while demonstrating a good tolerability profile with no body weight changes and normal serum chemistry. Development of therapeutic siRNA constructs can be hampered by off-target effects and cytokine induction. Substitution of unlocked nucleobase analogs (UNAs) at specific positions in the passenger and guide strands of siRNA confers high specificity with minimal to no off-target activity. UsiRNAs were successfully delivered to bladder or abdominal tumors when encapsulated in DiLA2- or SMARTICLES®-derived liposomes. DiLA2−encapsulated polo-like kinase 1 (PLK1) UsiRNA demonstrated 95% mRNA reduction and up to 70-fold tumor growth inhibition in a bladder cancer orthotopic mouse model and 40-70% mRNA inhibition and ∼80% reduction in tumor-specific bioluminescence in a malignant ascites mouse model following intravesical or intraperitoneal dosing, respectively. In an orthotopic liver tumor animal model, liposomal encapsulated miRNA mimetics provided ∼80% reduction in tumors compared to buffer control. Combinations of formulations and construct capitalize on the advantages of nucleic acid-based therapeutics for precise targeting and flexible delivery to challenging drug targets in cancer and other indications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 281. doi:1538-7445.AM2012-281
Abstract 2819: Temsirolimus inhibits colon cancer growth in vitro and in vivo
Authors: Kaneko Manabu , Nozawa Hiroaki , Tsuno Nelson H. , Kawai Kazushige , Sasaki Kazuhito , Hongo Kumiko , Hiyoshi Masaya , Sunami Eiji , Takahashi Koki , Kitayama Joji
Published: August 2012

Objective: Temsirolimus (CCI-779), an ester of the macrocyclic immunosuppressive agent sirolimus, is a cytostatic cell cycle inhibitor with antitumor properties. Temsirolimus inhibits the mammalian target of rapamycin (mTOR), a serine-threonine kinase involved in the initiation of mRNA translation, and has been shown to inhibit the growth of a variety of tumor cells. However, there have been only a few reports concerning the effect of temsirolimus on colon cancer cells. In the present study, we aimed to investigate the mechanisms of antitumor activity of temsirolimus in colon cancer cells in in vitro and in vivo studies. Methods: Two human colon cancer cell lines, CaR1 and LOVO, were treated with temsirolimus at a range of concentrations. Cell proliferation, cell cycle and apoptosis indices were evaluated to assess antitumor effects in vitro. The effect of temsirolimus on the proliferative activity was investigated by the MTS assay. The cell cycle was evaluated by flow-cytometry after staining of cell with PI. Apoptosis was analyzed by flow-cytometry after double-staining of cells with AnnexinV/PI. PI3K/AKT/mTOR pathway related proteins were measured by Western blot analysis. The in vivo effects of temsirolimus on tumor cell growth were examined using subcutaneous xenograft mouse model. Temsirolimus treatment (0, 0.1, 1, 10 mg/kg, 3 days/week, i.p.) was started when tumor reached approximately 100-200 mm3, and repeated three weeks. Tumor volume was calculated using the following equation: Volume (mm3) = Length x Width2 x 1/2. Results: Temsirolimus decreased the ability of colon cancer cells to proliferate in vitro in time and dose dependent manners. Furthermore, temsirolimus induced G1 cell cycle arrest, but did not induce apoptosis. Western blot analysis revealed that temsirolimus downregulated phospho-S6K, which phosphorylates the S6 protein of the 40S ribosomal subunit and leads to initiation of protein synthesis. In murine xenograft model, temsirolimus reduced tumor volume in a dose-dependent manner. After three weeks treatment, the average tumor volume in the vehicle group was about four times larger than that in the group receiving temsirolimus at a dose of 10mg/kg. Conclusions: Temsirolimus suppressed the proliferation of colon cancer cells in vitro through induction of G1 cell cycle arrest and downregulation of phospho-S6K. The tumor suppressive effect of the mTOR inhibitor was reproduced in an in vivo model. Our present findings indicate that mTOR signaling is a potential target of colon cancer, and the mTOR inhibitor temsirolimus may be a promising agent for colon cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2819. doi:1538-7445.AM2012-2819
Abstract 2824: Therapeutic potential of multi-kinase inhibitor sorafenib in osteosarcoma cells
Authors: Ko Yunmi , Lee Jun Ah
Published: August 2012

Osteosarcoma (OS) is the most common primary malignant bone tumor in childhood and adolescence. The introduction of multiagent chemotherapy has greatly improved the survival of patients with osteosarcoma, however, the improvement in survival has reached a plateau. Therefore, search for novel agents focused on different mechanisms is mandatory to further improve the survival of osteosarcoma patients. Sorafenib is the first oral multi-kinase inhibitor that directly blocks the auto-phosphorylation of several receptor tyrosine kinases (VEGFR1, 2 and 3, PDGFRα, c-Kit and RET) and inhibits Raf kinase isoforms (wild-type Raf1, and B-Raf and mutant b-raf V600E) activity to prevent tumour growth by anti-angiogenic, antiproliferative and/or pro-apoptotic effects. Here, we describe the effect of sorafenib in human OS cell lines on the proliferation and migration. And we also report combination effects with irradiation (IR) and cisplatin. We measured cell proliferation by MTT assay and used Annexin V and flow cytometry to assess whether sorafenib induce apoptosis in the OS cells. Next the migration of sorafenib-treated OS cells was determined by wound-healing assay and the synergistic effect between sorafenib and IR was evaluated using colony-forming assay. Also CI values of drug combinations of cisplatin and sorafenib in OS cells were calculated using Calcusyn software to assess whether sorafenib can be useful when used in conjunction with cisplatin. We found that sorafenib effectively inhibits cell proliferation, induced apoptosis, and prevents cellular migration. And our results demonstrate that the combination of two drugs, sorafenib and cisplatin, showed synergistic effects and colony-forming assay showed that sorafenib enhanced the radiosensitivity of OS cells. In conclusion, we found that sorafenib exerts therapeutic potentials in OS cells and suggests the possibilities of using sorafenib as a potential adjuvant therapy for the treatment of osteosarcoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2824. doi:1538-7445.AM2012-2824
Abstract 2834: Discovery of a novel class of prostaglandin e2 synthesis inhibitors with anti-tumor activity in colorectal cancer
Authors: Sells Earlphia , Medda Federico , Chang Hui-Hua , Gokhale Vijay , Hulme Christopher , Meuillet Emmanuelle J.
Published: August 2012

Prostaglandin E2 (PGE2), a key mediator of inflammation, is the most common prostanoid with various bioactivities and has been associated with many pathologies. Thus, inhibition of PGE2 synthesis and its action has been suggested in the treatment of inflammatory-associated diseases, including cancer. Microsomal Prostaglandin E2 Synthase-1 (mPGES-1) is an enzyme downstream of the cyclooxygenases (COXs) in the PGE2 biosynthesis pathway. mPGES-1, which is one of the three PTGES isoforms, is constitutively expressed in many cancers, including colon cancer. The potential role for this enzyme in tumorigenesis has been reported. Since inhibition of COXs lead to side effects due to the global reduction of other key prostaglandins, targeting downstream m-PGES-1 might be a better therapeutic strategy for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we have screened several libraries in silico and used published scaffolds exhibiting anti-inflammatory properties to initiate our study. As a result, 10 compounds were identified by molecular docking simulations using the reported crystal structure of mPGES-1. Subsequently a total of 32 analog compounds were synthesized or purchased and tested for their ability to inhibit COX-2 in vitro activity as well as for their ability to inhibit PGE2 production in an HCA-7 colorectal cancer cell line. We show that 16 of the 32 compounds reduced PGE2 synthesis by over 80% at 1 µM, with only one of these compounds inhibiting more than 30% of COX-2 activity at 5 µM in vitro. Furthermore, one compound, PGE0056 (EC50= 0.37 ± 0.06 µM), exhibited anti-tumor activity after 10 days at 100mg/kg i.p. in HCA-7 colon cancer xenograft mouse model. Following treatment with PGE0056, PGE2 production was reduced in the blood of scid mice. In summary, our data suggest that these new identified compounds exert anti-tumor activity at least in part through inhibition of the COX2/mPGES-1 enzymes within the PGE2 synthesis pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2834. doi:1538-7445.AM2012-2834
Abstract 2841: A simplified approach to measuring thymidine kinase activity in cells, tumors, and blood as a biomarker of tumor proliferative potential
Authors: Li Wenlin , Kang Xiaolin , Kindt Kristian E. , Zhang Cathy , Spilker Mary , Berlinski Pamela J. , Eisenbraun Michael , Yan Zhengming , Elliott Mark , Arbuckle J. Alan , Faria Morse , Karnes H. Thomas , O'Brien Peter J.
Published: August 2012

Understanding tumor size and growth is essential for preclinical oncology drug development, but costs and technical hurdles can prevent accurate, repeated measurements of cell proliferation and tumor growth. Many investigators instead measure the accumulation of nucleosides and analogs (e.g., [18F]-FLT; [3H]-Thy; BrdU) in cells and tissues, a manifestation of thymidine kinase 1 activity (TK1a). These methods can be costly and laborious, and can generate significant hazardous waste, promoting the use of even less-direct measures of cell division. Seeking further insights into the role of TK1 in cancer, we developed rapid, robust, non-isotopic methods that measure TK1a in tissue extracts and blood. In this approach, samples are exposed to FLT in vivo or in vitro, promoting its phosphorylation. Subsequently, nucleosides and their phosphorylated metabolites are isolated for analysis by a simple organic extraction. Using these methods, TK1a was measured in primary and transformed cells, and showed excellent comparability with established proliferation assays including 3H-thymidine incorporation, ATP generation, and fluorescent dye dilution flow cytometry. In drug screens, our method was as sensitive as commercial viability assays, and typically detected drug effects much sooner than those less-specific measures. We also demonstrated chemotherapy-induced alterations in TK1a in xenografted tumor tissue that were comparable to results obtained using radioisotopes. Finally, this approach was adapted to measure TK1a in blood samples. Consistent with published reports, circulating TK1a correlated well with tumor burden in solid tumor models and primary canine hematological cancer. These methods require only small sample volumes (<50μL serum or other biofluids, or a few thousand cells), allowing serial blood collection for monitoring tumor growth kinetics in rodents. These translational studies are underway, and should greatly inform our understanding of the role of TK1 in tumor responses to treatment in vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2841. doi:1538-7445.AM2012-2841
Abstract 2851: Cabozantinib (XL184) inhibits prostate tumor growth and bone lesion development
Authors: Dai Jinlu , Zhang Honglai , Karatsinides Andreas , Aftab Dana , Schimmoller Frauke , Keller Evan T.
Published: August 2012

Over 80% of advanced prostate cancers (PCa) metastasize to bone resulting in primarily osteoblastic bone lesions. Cabozantinib (CABO) is a multi-kinase inhibitor that targets MET and vascular endothelial growth factor receptor-2 (VEGFR2). Administration of CABO to men with PCa bone metastases has been shown to induce a marked resolution of lesions on bone scan as well as regression of measurable disease. Modulation of prostate specific antigen (PSA) appeared to be independent of changes in other parameters such as target lesions and bone scan. The current study was designed to determine mechanisms underlying CABO activity in PCa. Expression of MET and VEGFR2 or modulation of their activity were verified in PCa cell lines such as PC-3 or the pre-osteoblast cell line MC3T3-E1. To evaluate if CABO impacts PCa neoplastic phenotypes, PCa cell lines LNCaP, C4-2B and PC-3 were treated. CABO inhibited cell viability, migration and invasive ability and promoted apoptosis in LNCaP, C4-2B and PC-3 cells. To determine if CABO could directly modulate PSA expression, LNCaP (androgen dependent) and C4-2B (androgen independent) cells were incubated in the presence or absence of CABO. CABO increased both PSA and androgen receptor (AR) mRNA and protein expression in the LNCaP cells but did not modulate PSA expression in C4-2B cells. MC3T3-E1 and ST2 cells were treated with CABO to evaluate impact on osteoblastogenesis. CABO decreased cell viability and modulated osteocalcin levels in MC3T3-E1 and ST2 cells. ACE-1 canine osteoblastic PCa cells were injected into the tibia and treatment with CABO was started after tumors had developed to determine the impact on PCa growth in vivo. CABO decreased both ACE-1 tumor burden and tumor-induced osteoblastic activity in bone. In contrast, when PC-3 cells (osteolytic) were injected subcutaneously or into the tibiae of mice, which were treated with CABO or vehicle once tumors were established, CABO slowed the growth of PC-3 tumors in the soft tissue but not in bone. These findings indicate that CABO inhibits prostate tumor growth and tumor-induced bone lesion development through direct targeting of both PCa cells and osteoblasts. These studies provide a strong rationale to further elucidate the effect of CABO on osteoblastogenesis and PSA modulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2851. doi:1538-7445.AM2012-2851
Abstract 2856: Functional profile of BEZ-235 activity in human tumor primary culture microspheroids by ex vivo analysis of programmed cell death (EVA/PCD)
Authors: Nagourney Robert Alan , Bernard Paula J. , Francisco Federico R. , Evans Steven S.
Published: August 2012

Aberrant signaling through the phospho-inositol pathway via the gain of PI3K function or loss of PTEN is a hallmark of many malignancies. Drugs that inhibit this pathway offer novel therapeutic opportunities. The imidazoquinolone derivative, BEZ-235, reversibly inhibits signaling through PI3K & mTOR by competition at ATP binding sites. We used ex vivo analysis of programmed cell death (EVA/PCD) to examine the activity and clinical potential of BEZ 235 in human tumors isolated from 88 surgical tumor specimens. By interrogating drug effects in primary culture micro-spheroids, replete with vascular, stromal and inflammatory elements, the EVA/PCD platform can provide insights into cellular responses under native-state conditions. Lethal concentration 50% values (LC50's), interpolated from dose response curves, were used to compare the activity of BEZ-235 by diagnosis using modified Z-scores. Comparisons between BEZ activity and related inhibitors of PI3K (LY294002), mTOR (Everolimus) & AKT (Phen B15-kindly provided by Dr. Peter Houghton) were conducted by Pearson-Moment. By rank order, BEZ-235 activity revealed upper gastrointestinal, breast, NSCLC and hematological malignancies to have the most favorable profiles, while renal, ovarian, colon and sarcoma specimens fell in the more resistant range. Pearson moments revealed high correlation coefficients (R values) for LY294002=0.56 (P =0.001); Everolimus=0.51 (P= 0.005) & Phen B15=0.89(P= 0.005) all consistent with BEZ-235's known modes of action. As PI3K signaling is associated with inhibition of apoptosis, its up-regulation may confer collateral resistance to other stressors like growth factor withdrawal or cytotoxic drugs. Relationships between BEZ-235 and other classes of drugs are being examined to explore additional correlations and potential combination that could provide future therapeutic opportunities, as will be reported. Supported in part by the Vanguard Cancer Foundation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2856. doi:1538-7445.AM2012-2856
Abstract 2860: EphA2 and its phosphorylation is a key determinant of dasatinib sensitivity in endometrial cancer
Authors: Huang Jie
Published: August 2012

Background: Growing evidence points to EphA2 having bidirectional signaling to promote or inhibit tumorigenicity in ligand-independent and ligand-dependent manner. But the mechanistic and biological impact of this EphA2 dual switch function in the context of EphA2 targeted therapy in cancer is not known. Materials and Methods: The effects of dasatinib alone or in combination with paclitaxel on human endometrial cancer cell lines were examined using a series of in vitro experiments, including MTT, RPPA, Western blot, ELISA, and plasmid transfection (for inactivated or constitutively activated phosphorylation at EphA2-S897). In vivo, an orthotopic mouse model of endometrial cancer was utilized to identify the biological effects of dasatinib in orthotopic endometrial cancer models. Molecular markers for response prediction were identified by using ELISA, RPPA and immunohistochemistry. Results: In vitro, the effects of dasatinib treatment varied among the different endometrial cancer cell lines tested, (IC50 values ranged from 10 nM to10,000 nM) which was associated with varying levels of EphA2 and EphA2S897 phosphorylation, basal level of p-AKT and caveolin-1 in the EphA2 positive (Hec-1A, Ishikawa, and SKUT-2) and negative cells (SPEC-2). Moreover, in vivo experiments showed that dasatinib monotherapy or in combination with paclitaxel led to tumor inhibition over controls in the EphA2 positive Hec1A and Ishikawa murine models. Specifically, dasatinib treatment resulted in 48% and 71% reduction of tumor growth in these models, respectively (p<0.01). Combination of dasatinib and paclitaxel showed even greater efficacy (87% - 89% reduction in tumor growth, p<0.001). In the EphA2 negative SPEC2 murine models, dasatinib therapy resulted only in modest efficacy (31%, p<0.05), while the efficacy in combination with paclitaxel was better (75%, p<0.01). The antitumor effects of dasatinib were significantly associated with EphA2 and its phosphorylation status at S897, especially in the tumors with high expression of caveolin-1, as detected by RPPA, Western blot analysis and immunohistochemistry. Conclusion: The efficacy of dasatinib therapy in uterine cancer models depends on the status of the EphA2 and its activation of p-EphA2s897. These findings may have clinical implications for future dasatinib-based clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2860. doi:1538-7445.AM2012-2860
Abstract 2863: Development of an AKT1-specific synbody that inhibits MOLT-4 cell growth
Authors: Diehnelt Chris W.
Published: August 2012

Our group has developed bivalent peptide affinity ligands, called synbodies, to a number of proteins, including the serine / threonine kinase, AKT1. However, despite the high affinity of the synbody for AKT1 (KD = 1.5 nM), questions remain about the use of this synbody as a probe to modulate AKT1 activity within the cell due to the impermeability of most peptide ligands. In this presentation, we demonstrate that the AKT1 synbody penetrates mammalian cells and localizes with AKT1 in MOLT-4 acute lymphoblastic leukemia cells. The synbody specifically binds AKT1 but not AKT2 or AKT3 in the cytoplasm of MOLT-4 cells demonstrating a level of specificity typically only achieved by antibodies, which do not normally penetrate the cell. It was also found that after 24 hours of treatment, the synbody inhibits growth of MOLT-4 cells with an IC50 of 5 uM. FACS analysis revealed that the synbody induces apoptosis by altering the downstream activity of AKT1. We are currently confirming the mechanism of action of the synbody as well as preparing modifications to increase the activity and in vivo stability of the synbody.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2863. doi:1538-7445.AM2012-2863
Abstract 2864: Regulation and targeting of Fyn in cutaneous squamous cell carcinoma
Authors: Fenton Sarah , Denning Mitchell F.
Published: August 2012

Over 700,000 cases of cutaneous squamous cell carcinoma (cSCC) occur in the United States every year. Despite this high rate of diagnosis no therapies currently exist that target the molecular mechanism of tumorigenesis. Between 20 and 60% of these tumors contain mutated Ras; however targeting Ras with therapeutics has been unsuccessful in clinical trials. Expression of active H-Ras (G12V) in HaCaT cells (immortalized, non-transformed keratinocytes) results in upregulated expression of Fyn, a Src family kinase that is active in many malignancies and is involved in the elevated invasion/migration of Ras-transformed HaCaT cells. The specific objectives of this project include exploring the mechanism through which Ras activation increases Fyn levels and determining how high levels of Fyn promote cancer cell migration and survival. Using RT-qPCR we verified that Fyn expression is selectively elevated in cells with increased Ras activity, but levels of other Src family kinases (eg Src, Lyn, Yes, Blk, Lck) are not altered. To explore the transcriptional regulation of Fyn in Ras-transformed HaCaT cells we subcloned segments (−2 kb to −50 bp) of the human Fyn promoter in front of firefly luciferase and transfected the reporter constructs into HaCaT and HaCaT-Ras cells (transformed by constitutively active H-Ras). We identified a region −100 to −50 base pairs upstream of the transcription start site that is necessary for Fyn upregulation in HaCaT-Ras cells. The −80 to −65 region of the Fyn promoter contains putative GATA-1, GATA-2, GATA-3, CP2 or TATA transcription factor binding sites. We have also targeted Fyn in Ras-transformed cells using the Src family kinase inhibitor Dasatinib. Already used to treat chronic myelogenous leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia, Dasatinib is also in clinical trials to treat solid tumors. Treatment with varying concentrations of Dasatinib did not reveal greater changes in cell viability, cell cycle progression, apoptosis or autophagy in transformed HaCaT-Ras cells as compared to HaCaT cells. However, upon Dasatinib exposure HaCaT-Ras cells change dramatically in morphology, enhancing cell-cell contacts and reverting from a stellate appearance to the rounded morphology of untransformed HaCaT cells. Due to previous work verifying Fyn's role in promoting the invasion and migration of transformed keratinocytes, we propose that Fyn inhibition in keratinocytes with increased Ras activity will restore cell to cell contacts and provide a potential therapeutic target in the treatment of cSCCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2864. doi:1538-7445.AM2012-2864
Abstract 2870: p28, a cell penetrating peptide fragment of azurin, inhibits COP1 mediated ubiquitination of wild type and mutated p53, but does not alter intracellular levels of c-jun
Authors: Yamada Tohru , Christov Konstantin , Shilkaitis Anne , Bratescu Laura , Green Albert , Santini Simona , Bizzarri Anna Rita , Cannistraro Salvatore , Gupta Tapas K. Das , Beattie Craig W.
Published: August 2012

Amino acids (aa) 50-77 of azurin, p28, a 128 aa member of the cupredoxin family of copper containing redox proteins, isolated from the opportunistic pathogen Pseudomonas aeruginosa is a cell penetrating peptide (CPP) that preferentially penetrates a wide variety of human cancer cells. Exposure of p53wt, mut cancer cells to p28 produces a post translational increase in the level of p53, inhibition of the cell cycle at G2/M and apoptosis (Yamada et. al., Mol. Can. Therap., 8:2947-58, 2009). Computer simulation (ClusPro, GROMACS) and domain specific antibodies to p53 suggest p28 binds to motifs that span aa 110–146 in flexible L1 loop and 80 and 276 of the p53 DNA binding domain (DBD), respectively. Western and RT-PCR analyses of human breast cancer, MCF-7 (p53wt), MCF-7 derived MDD2 (p53dom/neg), MDA-MB-231 (p53mut), and melanoma cells, Mel-29 (p53wt), Mel-23 (p53mut) and Mel-6 (p53null) exposed to p28 for 72 hrs showed a significant increase in the levels of p53 and p21 in MCF-7, MDA-MB-231, Mel-29 and Mel-23 cells, while significantly reducing the level of FoxM1 over a 72hr exposure. The level of STMN1 was not altered suggesting the block at G2/M does not involve microtubules. p28 significantly decreased the level of the E3 ligase COP1 in p53wt, mut MCF-7, MDA-MB-231, Mel-29 and Mel-23, but not in p53null Mel-6 cells. In addition, p28 did not significantly decrease the level of E3 ligases TOPORS, Pirh2 or HDM2 which also ubiquitinate p53. The levels of p53, p21, FoxM 1, STMN1, COP1, TOPORS and Pirh2 either remained essentially at control levels (p53, p21) or were elevated (FoxM1, STMN1, COP1, TOPORS, Pirh2) at some point during a 72 hr exposure of p53dom/neg MDD2 cells to p28. In contrast, COP1 has also been reported to negatively regulate cell proliferation in a c-jun-dependent manner and not regulate intracellular levels of p53 (Migliorini et. al., J. Clin. Invest., 121: 1329-43, 2011). The p28 induced decrease in the level of COP1 in p53wt, mut cancer cells was not accompanied by a sustained increase in c-jun, in any cancer cell line, suggesting that COP1 regulates the level of p53 in cancer cells independent of any effect on c-jun. These data also suggest that COP1, but not TOPORS or Pirh2, also binds within this region of p53, and that COP1 is a major regulator of p53 activity. In sum, these data suggest that p28 binds to a specific motif within the p53 DBD where it inhibits COP1 mediated binding to and ubiquitination of p53.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2870. doi:1538-7445.AM2012-2870
Abstract 2873: Opposing effects of NF-κB inhibition during chemical lung carcinogenesis
Authors: Zaynagetdinov Rinat , Karabela Sophia P. , Psallidas Ioannis , Sherrill Taylor P. , Kairi Chrysoula A. , Cheng Dong-Sheng , Vassiliou Spyridoula , Gleaves Linda A. , Han Wei , Zakynthinos Spyros G. , Yull Fiona E. , Roussos Charis , Kalomenidis Ioannis , Blackwell Timothy S. , Stathopoulos Georgios T.
Published: August 2012

Since recent evidence indicates a requirement for epithelial NF-κB signaling in lung tumorigenesis, we investigated the impact of the NF-κB inhibitor bortezomib on lung tumor promotion and growth. We used an experimental model in which wild-type mice or mice expressing an NF-κB reporter received intraperitoneal urethane (1 g/kg) followed by twice-weekly bortezomib (1 mg/kg) during distinct periods of tumor initiation/progression. Mice were serially assessed for lung NF-κB activation, inflammation, and carcinogenesis. Short-term proteasome inhibition with bortezomib did not impact tumor formation, but retarded the growth of established lung tumors in mice via effects on cell proliferation. In contrast, long-term treatment with bortezomib resulted in significantly increased lung tumor number and size. This tumor-promoting effect of prolonged bortezomib treatment was associated with perpetuation of urethane-induced inflammation and chronic up-regulation of interleukin-1α and proinflammatory C-X-C motif chemokine ligands (CXCL) 1 and 2 in the lungs. In addition to airway epithelium, bortezomib inhibited NF-κB in pulmonary macrophages in vivo, presenting a possible mechanism of tumor amplification. In this regard, RAW264.7 macrophages exposed to bortezomib showed increased expression of interleukin-1α, CXCL1, and CXCL2. In conclusion, although short-term bortezomib may exert some beneficial effects, prolonged NF-κB inhibition accelerates chemical lung carcinogenesis by perpetuating carcinogen-induced inflammation. Inhibition of NF-κB in pulmonary macrophages appears to play an important role in this adverse process.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2873. doi:1538-7445.AM2012-2873
Abstract 2875: Meta-iodo-benzylguanidine (MIBG) is a MYCN/MYC destabilizer that specifically targets neural crest derived tumors
Authors: Wang Stephanie , Maloney Nolan , Hsiao Ruth , Limpar Mariko , Ikegaki Naohiko , Tang Xao X.
Published: August 2012

Melanoma and malignant pheochromocytoma are known for high MYC expression. MYCN amplification and its over-expression are associated with the worst neuroblastoma disease outcome. Meta-iodo-benzylguanidine (MIBG) is a norepinephrine analogue and a mitochondrial respiration inhibitor. 131I-MIBG has been used for scintigraphic detection of neural crest derived tumors (neuroblastoma, pheochromocytoma and melanoma) that specifically uptake MIBG by norepinephrine transporters (NET). 131I-MIBG is also used for targeted radiotherapy of neuroblastoma and pheochromocytoma. Non-radiolabeled MIBG had been reported to be cytotoxic to neuroblastoma cells in vitro and in vivo. However, the molecular mechanism of its growth suppressive effect was not elucidated. Our previous studies showed that FCCP, a well-known mitochondrial respiration inhibitor, destabilized MYCN and MYC in neuroblastoma cells and caused growth suppression. In this study we confirmed that MIBG suppressed growth of neuroblastoma cell lines (SKNBE(2)C, IMR5, Nb69, and SKNAS), and its growth suppressive effect in each cell line correlated with NET expression in these cells. In addition, MIBG suppressed growth of neuroblastoma stem cells derived from SKNBE(2)C. MIBG treatment of SKNBE(2)C and SKNAS resulted in an increase expression of growth suppressive gene (GADD45B) and genes encoding for biomarkers of favorable neuroblastoma (CD44, EPHB6, EFNB2, EFNB3, NTRK1). Treatment of MYCN amplified (SKNBE(2)C, IMR5) and non-MYCN amplified (Nb69, SKNAS) neuroblastoma cells with MIBG resulted in marked reduction of MYCN and MYC expression, respectively in dose and time dependent fashions. Collectively, our studies suggest that MIBG not only targets neural crest derived tumors, but also is a MYCN/MYC destabilizing agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2875. doi:1538-7445.AM2012-2875
Abstract 2885: Urokinase plasminogen activator system targeted delivery of arsenic trioxide loaded nanobins as a novel ovarian cancer therapeutic
Authors: Zhang Yilin , Swindell Elden P. , Ahn Richard W. , Mitra Anirban , Mazar Andrew P. , O'Halloran Thomas V. , Kenny Hilary A. , Lengyel Ernst
Published: August 2012

That the urokinase plasminogen activator receptor (uPAR) system is constitutively expressed in primary and metastatic ovarian cancer regardless of grade or stage, while it is only weakly expressed in normal tissues, identifies it as a possible therapeutic target. Taking advantage of the tumor-specific expression of uPAR, a nanoscale liposome (nanobin, NB) was coupled with an antibody (ATN 291) against the uPAR ligand, uPA, to facilitate receptor-mediated, targeted drug delivery. The nanobin was loaded with arsenic trioxide (As2O3, in short As), since our previous results had demonstrated effective tumor uptake and induction of tumor cell apoptosis using As loaded nanobins in an orthotropic model of human triple negative breast cancer. We hypothesized that the ATN 291-conjugated nanobin (NB-uPA Ab) would allow specific cellular internalization and increase the therapeutic index of As in ovarian cancer cells. Using flow cytometry and fluorescence microscopy, we measured the cellular uptake of NB(Calcein)-uPA antibody (Ab) in HeyA8, ovarian cancer cells. The data showed a dose-dependent pattern of internalization with maximum internalization at 24 hours. Furthermore, NB-uPA Ab showed elevated cellular uptake when compared to non-targeted NB. More importantly, delivery of the targeted nanobins is uPA/uPAR dependent. High doses of ATN-291 or scuPA inhibited the cellular binding of NB-uPA Ab to HeyA8 cells. Moreover, down-regulation of uPAR expression via RNA interference in ovarian cancer cells inhibited the cellular uptake of the NB-uPA Ab. The cell viability of HeyA8 cells was significantly reduced following treatment of both non-targeted NB(As) and NB(As)-uPA Ab when compared to empty NB treatment as determined by a cytotoxicity assay. However, the cytotoxicity of NB(As)-uPA Ab is 2.1-fold more potent than the non-targeted NB(As). Taken together, these data indicate that the uPA Ab conjugated nanobins improved drug delivery and enhanced the anti-cancer effects of As, demonstrating the utility of targeting the uPA/uPAR system in ovarian cancer chemotherapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2885. doi:1538-7445.AM2012-2885
Abstract 3013: Role of prostanoid signaling in lung cancer
Authors: Pamarthi Pavankumar , Savai Rajkumar , Pullamsetti Soni Savai , Seeger Werner , Grimminger Friedrich , Schermuly Ralph
Published: August 2012

RATIONALE: An increasingly large body of evidence indicates that PGE2 promotes tumor growth by stimulating EP4 receptor signalling with subsequent enhancement of cellular proliferation, metastasis and suppression of immune responses. However, the role of other EP receptors is not known. OBJECTIVE: We hypothesized that prostanoid receptors other than the EP4 receptor are involved in progression of Lung cancer. METHODS: We analyzed the expression profile of EP receptors in lung adenocarcinoma cell lines A549 and H1299 by using Real Time PCR, western blotting and immunofluorescence. All four EP receptors are getting expressed in both the cell lines. Both L-161982 (EP4 receptor specific antagonist) and SC-51322 (EP1 receptor specific antagonist) inhibited proliferation of both cell lines as assessed by thymidine incorporation assay. EP2 and EP3 receptors have not shown inhibitory affects in proliferation assays. SC-51322 has not decreased cAMP levels in A549 and decreased intracellular calcium levels. SC-51322 has inhibited pERK levels as shown in the western blots. In addition, SC-51322 inhibited migration of A549 cells in transwell migration assay. CONCLUSION: We conclude that EP1 receptor may play a role in lung cancer progression and metastasis. Finding out downstream molecules to EP1 receptor will give us a better understanding of this pathway in lung cancer and can provide therapeutic opportunities.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3013. doi:1538-7445.AM2012-3013
Abstract 3049: Development of novel survivin inhibitors to target mitotic machinery in cancers
Authors: Cooley James V. , Chettiar Somsundaram , Park In-Hee , Bhasin Deepak , Chakravarti Arnab , Kai Li Pui , Li Chenglong , Jacob Naduparambil K.
Published: August 2012

Survivin, a member of the inhibitor of apoptosis (IAP) protein family has essential roles in cell division and inhibition of apoptosis. Over-expression of survivin is observed in most human cancers, but survivin expression is not detectable in non-proliferating normal adult tissues. In dividing cells, survivin is detected as an integral component of the chromosomal passenger complex (CPC). In the CPC, survivin is organized as a homodimer, associated with Aurora B-kinase and plays a critical role in the recruitment of a number of proteins to the kinetochore and centromere, which are essential for mitotic progression. Several clinical correlative studies in cancer patients have shown that elevated levels of survivin decreases overall survival and leads to resistance to radiation and chemotherapeutic treatments. Thus, disruption of functional survivin along its dimer interface is hypothesized to inhibit the proliferation of cancer cells by further sensitizing them to therapeutic agents and radiation. Recently, through HTS-NMR and AS/MS affinity based screenings, compound 1 (C1) was identified to bind to the dimerization interface of survivin monomers. Further development of this compound was accomplished by computational modeling of inhibitor interactions along the dimerization interface, which has lead to the design of a panel of promising compounds analogous to compound 1. Two of the most potent inhibitors included LLP3 and LLP9, which showed significant levels of activity between 50 and 100 nM. In proliferating human umbilical vein endothelial cells (HUVEC) as well as in prostate cancer cells (PC3) cells, the compounds caused a delay in mitotic progression and major mitotic defects. These cells expressed physiological levels of GFP-tagged survivin, which were used for visualization in automated fluorescent time-lapse videomicroscopy to investigate how the survivin dimerization inhibitors affected cell cycle progression. The same microscopy methods were used for understanding the specificity and activity of each respective compound. The investigation of cells treated with each of the compounds allowed for resolution of subcellular level effects on mitotic progression and CPC organization/movement during mitosis. The qualitative microscopy data was enumerated in order to quantify effects the compounds had on cells. The cells were enumerated in triplicate and tracked for changes in their proliferation, cell death, changes in ploidy, and other mitotic defects. The relationships between structure and activity of the compounds will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3049. doi:1538-7445.AM2012-3049
Abstract 3051: CDC28 protein kinase subunit 1B (CKS1B) is a potential therapeutic target in esophageal adenocarcinomas overexpressing the CDK inhibitor p21Cip1/Waf1
Authors: Ismail Amin , Bandla Santhoshi , Toia Liana , Godfrey Tony
Published: August 2012

Background: The incidence of adenocarcinoma of the esophagus and gastroesophageal junction (EAC) has grown faster than any tumor type in the United States and westernized countries, outpacing the next closest cancer by almost 3 times. This rising incidence of EAC is associated with the increasing prevalence of obesity and gastroesophageal reflux disease (GERD). Due to the limited effect of current chemotherapy for this cancer, the search for new therapeutic targets is disparately needed. Chromosomal amplification is one mechanism by which cancer cells acquire and maintain neoplastic phenotype and genes that are amplified and overexpressed may represent potential therapeutic targets. Methods: DNA and RNA from a cohort of 116 patients were analyzed using SNP and mRNA expression microarrays respectively. Gene amplification was explored for correlation with expression and patient survival. Results: 25% of tumors harbor amplification at chromosome 1q which is significantly associated with poor survival. Although the amplicon is non-focal spanning almost the entire arm of chromosome 1q, only a subset of amplified genes (including The CDC28 protein kinase subunit 1B, CKS1B) are significantly overexpressed. CKS1B is an auxiliary protein required for ubiquitination and cytosomal degradation of the CDK inhibitors p27Kip1 and p21Cip1/Waf1 by the SCFSKP2 E3 ubiquitin ligase complex. Both p27Kip1 and p21Cip1/Waf1 inhibits CDK2/Cyclin E complex during the G1/S cell cycle transition. The endogenous protein expression of p27Kip1 is low among EAC cell lines. However the expression of p21Cip1/Waf1 is differential being very high in a subset of cells and low or undetectable in others. Knocking down CKS1B by siRNA in six EAC cell lines results in accumulation of p27 Kip1 with little or no effect on p21Cip1/Waf1 expression. CKS1B knockdown inhibits monolayer cell proliferation only in cells with high endogenous expression of p21Cip1/Waf1. Conclusion: We suggest that the combined action of accumulation of p27 Kip1 and the endogenously highly expressed p21Cip1/Waf1 triggers the response to CKS1B knockdown in EAC cell lines. We thus propose the potential use of CKS1B as a novel therapeutic target in esophageal adenocarcinomas with high p21Cip1/Waf1 expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3051. doi:1538-7445.AM2012-3051
Abstract 3053: Identification of a novel cytoplasmic role for MTDH/AEG-1 in chemoresistance as a RNA binding protein
Authors: Meng Xiangbing , Zhu Danlin , Wang Xinjun , Yang Shujie , Zhang Yuping , Xiong Zhi , Leslie Kimberly K.
Published: August 2012

Overexpression of metadherin (MTDH/AEG-1) has been documented in many solid tumors and is implicated in metastasis and chemoresistance. MTDH has been detected at the plasma membrane as well as in the cytoplasm and nucleus, and the function of MTDH in these locales remains under investigation. In the nucleus, MTDH acts as a transcription co-factor to induce expression of chemoresistance-associated genes. However, MTDH is predominantly cytoplasmic in solid tumors, and this localization has been shown to correlate with poor prognosis. In this study, we used endometrial cancer cells as a model system to define a new role for MTDH in the cytoplasm. First, MTDH was primarily localized to the cytoplasm in endometrial cancer cells, and the N-terminal region of MTDH was required to maintain cytoplasmic localization. Depletion of cytoplasmic MTDH in endometrial cancer cells restored chemosensitivity to various DNA damaging agents including mitomycin C (MMC) and doxorubicin. Next, we identified novel binding partners for cytoplasmic MTDH, including ribosome protein RPL4, ribosome biogenesis-related protein NPM1, and a component of the RNA induced silencing complex SND1. Association of MTDH with these cytoplasmic proteins was disrupted by nuclease treatment, demonstrating that nucleic acid is required for the interaction of cytoplasmic MTDH with these RNA metabolism and processing proteins. Furthermore, RNA binding protein-chromatin IP (RIP-chIP) analysis demonstrated that MTDH interacted with multiple mRNAs, and treatment with the PI3K and mTOR dual inhibitor BEZ235 altered the pattern of mRNAs that associate with MTDH. Knockdown of MTDH promoted an increase in protein levels of some MTDH-associated mRNAs. Finally, increased stress granule formation was observed in MTDH knockdown cells in response to heat shock, suggesting that one mechanism by which MTDH promotes chemoresistance is prevention of stress granule formation. Taken together, our data identify MTDH as an active RNA binding protein in the cytoplasm of cancer cells. Our findings suggest that the RNA binding properties of cytoplasmic MTDH serve to regulate genes involved in the drug resistance and metastasis of tumors and highlight MTDH as an ideal target for future drug development. Acknowledgements: This work was supported by NIH Grant R01CA99908 to K.K.L., and in part by the Department of Obstetrics and Gynecology Research Development Fund, the Institutional Research Grant Number IRG-77-004-31 from the American Cancer Society to XM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3053. doi:1538-7445.AM2012-3053