bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2262: Contribution of autophagy in anticancer immune response induced by chemotherapy
Authors: Sukkurwala Abdul Qader , Michaud Mickaël , Martins Isabelle , Kroemer Guido
Published: August 2012

Antineoplastic chemotherapies are particularly efficient when they elicit immunogenic cell death, thus provoking an anticancer immune response. We demonstrate that autophagy, which is often disabled in cancer, is dispensable for chemotherapy-induced cell death but required for its immunogenicity. In response to chemotherapy, autophagy-competent, but not autophagy-deficient, cancers attracted dendritic cells and T lymphocytes into the tumor bed. Suppression of autophagy inhibited the release of adenosine triphosphate (ATP) from dying tumor cells. Conversely, inhibition of extracellular ATP-degrading enzymes increased pericellular ATP in autophagy-deficient tumors, re-established the recruitment of immune cells, and restored chemotherapeutic responses, but only in immunocompetent hosts. Thus, autophagy is essential for the release of immunogenic ATP from dying cells, and increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled.

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 2262. doi:1538-7445.AM2012-2262
Abstract 2264: Autophagy inhibition sensitizes the growth inhibitory effects of propolin C but not propolin G on human lung adenocarcinoma A549 cells
Authors: Hsu Hui-Chen , Wang Hsiao-Feng , Chen Yu-Fen , Chen Yue-Wen , Chen Wei-Jung , Tsai Meng-Feng
Published: August 2012

Propolins, prenylflavanones are isolated and characterized from Taiwanese propolis, have been shown inhibits certain cancer cell lines growth and induced apoptosis. In our previous study, Taiwanese propolis, propolin C and porpolin G, could promote LC3B increased within 6 hours treatment, moreover, and AVOs (acidic vesicular organelles) significantly increased by AO (Acridine orange) staining. The autophagy induced by propolin C or propolin G is accompanied by the inhibition of the PI3K/Akt/mTOR signaling pathway. Flow cytometry assay showed that sub-G1 phase increased significantly in propolin G treatment but not in propolin C treatment. Autophagy pharmacological inhibitors, 3-methyladenine and Chloroquine were used to test whether autophagy blockade could lead to enhance cell death. The results showed that only autophagy inhibitor chloroquine effectively enhanced apoptosis induced by propolin C but not propolin G in A549 cells. The expression of pERK and pAkt was suppressed dramatically by autophagy inhibitor in combination with propolin C. Ectopic expression of mutant Akt (constitutive active) in A549 cells abolished chloroquine sensitization effect of propolin C. Together, autophagy serves a protective role in propolin C-mediated cytotoxicity, and autophagy modulators may be used as adjunctive therapeutic agents for propolin.

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 2264. doi:1538-7445.AM2012-2264
Abstract 227: Overexpression of EphB1 leads to Stat3 phosphorylation
Authors: Gechtman Zev
Published: August 2012

The Receptor Tyrosine Kinase (RTK) family comprises some important disease drivers. Over-expressed or mutated RTKs in disease may lead to aberrant phosphorylation of intracellular signaling molecules. Therefore it is desirable to monitor simultaneously the activation levels of RTKs and signaling nodes. Here we used a multiplexed antibody array to monitor the phosphorylation of 28 RTKs and 11 intracellular signaling molecules simultaneously. We have observed a strong signal on a spot corresponding to phospho-STAT3 Tyr705 with lysates derived from CHO cells transiently over-expressing EphB1. The strong Phospho-STAT3 signal was not seen in non-transfected cells or in lysates made form cells transfected with several other Eph family members. Western blotting with phospho-specific antibodies showed an increase in STAT3 phosphorylation on Tyr705 in CHO cells upon EphB1 ectopic expression. The amounts of Tyr705 STAT3 phosphorylation positively correlated with the levels of EphB1 protein. Co-precipitation experiments showed that EphB1 and STAT3 interact both in CHO cells and MCF-7 breast cancer cells. Phosphorylated JAK2 was not found in these immuno-precipitates suggesting an involvement of another tyrosine kinase in STAT3 phosphorylation in cells over-expressing EphB1. Consistently with these observations, a potent JAK1 and JAK2 inhibitor INCB018424 inhibited the basal but not the EphB1-induced STAT3 Tyr705 phosphorylation in transfected CHO cells. These results suggest that EphB1 over-expression may bypass the requirement in Jak kinase and lead to STAT3 activation either directly or through another receptor-tethered kinase.

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 227. doi:1538-7445.AM2012-227
Abstract 2274: Docosahexaenoic acid-induced cell death may be related to inhibition of mTOR through AMPK activation and PI3K/Akt inhibition in human non-small cell lung cancer cells
Authors: Lim Kyu , Kim Nayeong , Jing Kaipeng , Jeong Soyeon , Shin Soyeon , Oh Hyerim , Jo Eun-Ah , Heo Jun-Young , Park Ji-Hoon , Seo Kang-Sik , Song Kyoung-Sub , Han Jeongsu , Park Jong-Il , Park Seung-Kiel , Kweon Gi-Ryang , Yoon Wan-Hee , Hwang Byung-Doo
Published: August 2012

Over 80% of the lung cancer patients have non-small cell lung cancer (NSCLC). Although the anticancer mechanisms of omega-3 polyunsaturated fatty acids (≥3-PUFAs) have been reported in several cancers, it is still unclear in lung cancer. In this study, we have identified a novel anticancer effect of docosahexaenoic acid (DHA), a α3-PUFAs, on NSCLC. DHA induced cytotoxicity and morphology change in A549 and H1299 NSCLC cells and was confirmed that apoptosis and autophagy are responsible for the cytotoxicity induced by DHA. DHA increased AMPK activity and inhibited PI3K/Akt signaling as well as mTOR signaling molecules. Knockdown AMPK using small interfering RNAs specific for AMPK and overexpression Akt significantly enhanced the mTOR activity and attenuated the cell death caused by DHA treatment, indicating that DHA induces NSCLC cell death via the AMPK- and Akt-regulated mTOR inactivation. We also confirmed this effect of α3-PUFAs in vivo using Fat-1 transgenic mice that are capable of producing α3-PUFAs. When mouse lung cancer LLC cells were subcutaneously implanted into Fat-1 mice, the growth of tumor was markedly inhibited with decrease the level of p-Akt as well as increases in TUNEL-positive staining cells and autophagic levels, compared to wild-type mice. Taken together, these data suggest that the apoptosis and autophagy induced by DHA may be related to mTOR inhibition through AMPK activation and PI3K/Akt inhibition in NSCLC. Therefore, utilization of DHA may represent a potential effective therapy for the chemoprevention and treatment of human non-small cell lung cancer. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (2011-0013263 and 2011-0006232)]

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 2274. doi:1538-7445.AM2012-2274
Abstract 2275: A novel heat shock protein 90 inhibitor, NVP-AUY922, enhances the antitumor effect of imatinib via autophagy on imatinib-resistant CML cells
Authors: Osato Yoichi , Yokoyama Tomohisa , Hayabe Hiroko , Miyasato Akime , Akashi Takao , Komatsu Seiichiro , Tauchi Tetsuzo , Ohyashiki Kazuma
Published: August 2012

The BCR-ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, has revolutionized the treatment of CML, however, the development of TKI-resistance caused in approximately 10-25% of cases by mutation in the BCR-ABL kinase domain. Therefore, novel therapeutic strategies are needed to address the emerging problem of imatinib-resistant CML. Recently, the heat shock proteins of 90 kDa (Hsp90) have emerged as targets of interest in cancer therapy. NVP-AUY922 (Novartis) is a novel 4, 5-diaryloxazole ATP-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and delete the levels of HSP90 client protein including BCR-ABL. We have recently reported that combined treatment of NVP-AUY922 and BCR-ABL TKI exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant form of BCR-ABL-expressing cells (Oncogene 30, 2789-97, 2011). However, the mechanism of programmed cell death (PCD) is not yet completely understood. In the present study, we examined the mechanism of PCD induction by the combined treatment of NVP-AUY922 and imatinib on wild-type (WT) and mutant forms of BCR-ABL Baf-3 cells (E255K and T315I). Imatinib-mediated inhibitory effect was observed on WT cells, however, E255K and T315I cells were less sensitive to imatinib than WT cells. BCR-ABL mutant T315I cells were the most resistant to imatinib. On the other hands, NVP-AUY922 was synergistically sensitized tumor cells to imatinib on both WT and imatinib-resistant Baf-3 cells. Activity of caspase-3/7 was increased by combined treatment with imatinib and/or NVP-AUY922 on WT cells, indicating induction of apoptosis. In contrast, apoptosis induced by combined treatment with imatinib and/or NVP-AUY922 was significantly suppressed, although the inhibition of cell growth was still detectable on E255K and T315I cells. May-Giemsa staining revealed that many vacuoles were detected in the cytoplasm by combined treatment with imatinib and NVP-AUY922 on E255K and T315I cells, indicating the morphologic features of autophagy. We are currently conducting autophagy-detection assay and determining autophagic signaling pathways. Our findings indicate that NVP-AUY922 enhances the antitumor effect of imatinib, through activation of autophagy on imatinib-resistant CML cells. These data strongly suggest that combined treatment of imatinib and NVP-AUY922 is a novel approach for the treatment of patients with imatinib-resistant CML.

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 2275. doi:1538-7445.AM2012-2275
Abstract 228: PAK1 kinase promotes cell motility through CRK serine phosphorylation in non-small cell lung cancer cells
Authors: Mortazavi Fariborz (Fred) , Rettig Matthew
Published: August 2012

The role of c-Crk (CRK) in promoting metastasis is well described but the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have noticed that CRK serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK serine 41 phosphorylation in the down-regulation of p120-catenin and cell motility in NSCLC cells. We prepared A549 cell lines stably expressing phosphomimetic and phosphodeficient CRK serine 41 constructs. A549 cells expressing phosphomimetic CRK seine 41 demonstrated a more aggressive behavior in would healing assay and on the contrary, expression of phosphodeficient CRK serine 41 in A549 cells reduced cell motility. A549 cells expressing phosphomimetic CRK serine 41 had lower p120-catenin mRNA and promoter activity and vice versa. In addition, we provide evidence that PAK1 mediates CRK serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level and also decreased cell motility in A549 and H157 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK serine 41. In summary, our data provide evidence for the role of PAK1 in promotion of cell motility and down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.

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 228. doi:1538-7445.AM2012-228
Abstract 2282: Targeting oncogenic NDRG4: A promising strategy for meningioma treatment
Authors: Kotipatruni Rama Prasadarao , Ren Xuan , Vanderwaal Robert , Thotala Dinesh Kumar , Jaboin Jerry
Published: August 2012

Meningiomas are the second most common brain tumors, and 20-30% of these tumors are aggressive. These aggressive subtypes are characterized by the capacity for invasion of normal brain with frequent and destructive recurrence patterns. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for this group or cancer patients. We have recently identified the N-myc down regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningioma, and have proposed to study its role in cell survival, invasion/migration and angiogenesis. We utilized shRNA-containing Lentiviral plasmids to downregulate NDRG4 mRNA and protein expression in two high grade meningioma cancer cell lines, IOMM-Lee and CH157MN. Downregulation resulted in reduction in cell survival, and G2-M cell cycle arrest. Utilizing DNA laddering and Annexin V/APC flow cytometry, we determined that the predominant form of cell death was apoptosis. NDRG4 downregulation also decreased cellular invasion and migration, as determined by a number of assays, including: spheroid migration, linear and radial wound healing assays, Boyden chamber matrigel invasion and 3D invasion assays. To determine the effect of NDRG4 depletion on angiogenesis, we utilized immortalized brain endothelial cells, bEnd.3 cells. We treated the bEnd.3 cells with conditioned media from the NDRG4 depleted IOMM-Lee and CH157MN cells, and the ability to form bEnd.3 capillary-like tubes, to proliferate and to invade was abrogated. NDRG4 is not overexpressed in bEnd.3 cells, and direct NDRG4 depletion had no effect on the cells. This study is significant as it is the first to demonstrate the role of NDRG4 in meningioma tumor biology. NDRG4 is involved in modulating cell proliferation, invasion, migration and angiogenesis in meningioma, and may play a valuable role as a molecular target in its 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 2282. doi:1538-7445.AM2012-2282
Abstract 2283: β-catenin silencing induces both apoptotic and autophagic cell death through LKB1/AMPK signal in head and neck cancer cells
Authors: Chang Hyo Won , Moon So Young , Kim Hyo Jung , Lee Myung Jin , Kim Mi Ra , Park Jung Je , Seo Ji Hyun , Kim Seong Who , Kim Sang Yoon
Published: August 2012

β-Catenin, a multifunctional protein involved in key molecule of Wnt signaling transduction, has been well known to regulate cell proliferation, differentiation, cell-cell adhesion, survival, and apoptosis as well as plays an important role in human tumorigenesis. Our previous report showed that Wnt/β-catenin pathway controls radiosensitivity of head and neck caner (HNC) cell via cyclooxygenase-2-mediated Ku expression. However, the specific molecular mechanisms of β-catenin in tumor cell survival and death are still not fully understood, so in the present study we attempted to elucidate the mechanism of cell death in HNC cell by studying how β-catenin silencing regulates cell death through KB1/AMPK signaling. We performed β-catenin knockdown in HNC cell by using small interfering RNA. Its effect on the cell viability was confirmed by observation of cell morphologic change, electron microscopy, MTT and FACS analysis. Also, we confirmed the expression of several proteins corresponding signal pathway by western blot analysis. β-catenin silencing significantly induced G1 cell-cycle arrest and also increased the expression of Bax and the active form of caspase-3, and decreased the expression of Bcl-2, demonstrating the sequential activation of apoptotic cascades following treatment with β-catenin siRNA in HNC cell. Interestingly, β-catenin silencing induced autophagy as well as apoptosis. We confirmed that the numbers of autophagic vacuoles and the expression of LC3-II increased in cells treated with β-catenin siRNA. This is probably because LKB1-dependent AMPK is activated by β-catenin silencing in HNC cell. Activated AMPK, in β-catenin silenced AMC-HN-3 cells, causes a G1 cell cycle arrest by phosphorylated p53 as well as inhibited protein synthesis by suppressing mTOR activity. Also, Treatment with LKB1 siRNA or compound C in AMC-HN-3 cells preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, our data show that β-catenin silencing induces cell death through LKB1/AMPK signal, and as a result, apoptosis and autophagy are induced together in HNC cell, suggesting that a novel interaction between β-catenin and LKB1/AMPK signaling pathway occurs. Our study provides new insights into understanding the cellular and molecular mechanisms involving β-catenin silencing-induced cell death.

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 2283. doi:1538-7445.AM2012-2283
Abstract 2285: Inhibition of autophagy and tumor growth in colon cancer by miR-502
Authors: Zhai Haiyan , Song Bo , Xu Xiao , Zhu Wei , Ju Jingfang
Published: August 2012

Autophagy is a catabolic process that allows cellular macromolecules to be broken down and recycled as metabolic precursors. The influence of non-coding microRNAs (miRNAs) in autophagy has not been explored in colon cancer. In this study, we discovered a novel mechanism of autophagy regulated by hsa-miR-502-5p (miR-502) by suppression of Rab1B, a critical mediator of autophagy. A number of other miR-502 suppressed mRNA targets (e.g. DHODH) were also identified by microarray analysis. Ectopic expression of miR-502 inhibited autophagy, colon cancer cell growth, and cell cycle progression of colon cancer cells in vitro. miR-502 also inhibited in vivo colon cancer growth in a mouse tumor xenografts model. In addition, the expression of miR-502 was regulated by p53 via a negative feedback regulatory mechanism. The expression of miR-502 was down-regulated in colon cancer patient specimens compared to the paired normal control samples. These results suggest that miR-502 may function as a potential tumor suppressor and therefore be a novel candidate for developing miR-502 based therapeutic strategies.

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 2285. doi:1538-7445.AM2012-2285
Abstract 2288: Genetic and epigenetic inactivation of miR-31 in EBV-associated nasopharyngeal carcinoma
Authors: Cheung Chartia Ching-Mei , Chung Grace Tin-Yun , Lun Samantha Wei-Man , Choy Kwong-Wai , To Ka-Fai , Lo Kwok-Wai
Published: August 2012

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignant cancer prevalent in Southern China and South-eastern Asia. In this study, we aim to elucidate the fundamental role of aberrant expressed microRNAs (miRNA) in NPC tumorigenesis. By comparison of miRNA expression profiles in NPC tumor lines with non-malignant nasopharyngeal epithelial cell, 119 differentially expressed miRNAs were identified in EBV-associated NPC. Among the 58 microRNAs downregulated in NPC, transcriptional silencing of miR-31 was consistently found in both NPC tumor lines and primary tumors. Expression of miR-31 was detected in none of 6 EBV-positive tumor lines and 38 microdissected primary tumors, but in all normal nasopharyngeal epithelia. Interestingly, miR-31 is located at 0.5 Mb telomeric to CDKN2A at 9p21.3, which is commonly homozygous deleted in NPC. Homozygous deletion of both miR-31 and CDKN2A locus was confirmed in 2 tumor lines. In the 4 tumor lines with intact miR-31, hypermethylation of 5′CpG islands was detected by bisulfate sequencing and MSP analysis. Re-expression of miR-31 was demonstrated in the EBV-positive NPC cell line C666-1 treated with 5-aza-2′-deoxycytidine. The findings suggested that homozygous deletion and promoter methylation are the major mechanisms for transcriptional silencing of miR-31 in NPC. By microarray and bioinformatic analysis, we have identified a number of putative targets of miR-31. Among these candidates, we have proven that miR-31 targeted FIH1 and MCM2 expression in NPC. Ectopic expression of miR-31 in NPC cells resulted in repression of FIH1 and MCM2 protein. Finally, we have demonstrated that restoration of miR-31 or knockdown of FIH1 expression significantly suppressed cell proliferation of C666-1. Our findings indicated that miR-31 is a NPC-associated microRNA which negatively regulates cell growth by repressing FIH1 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 2288. doi:1538-7445.AM2012-2288
Abstract 2300: MicroRNA-101 (miR-101) posttranscriptionally regulates the expression of EP4 receptor in colon cancers
Authors: Onyeagucha Benjamin C. , Chandramouli Anupama , Mercado-Pimentel Melania E. , Shahin Nisreen Abu , LaFleur Bonnie J. , Heimark Ronald L. , Bhattacharyya Achyut K. , Nelson Mark A.
Published: August 2012

The predominant product of cyclooxygenase (COX-2) in the colon, prostaglandin (PG) E2 promotes carcinogenesis. Expression of the PGE2 receptor EP4 is upregulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs. A bioinformatics search revealed a conserved target site for miR-101 within the EP4 receptor-3′ UTR. In both colorectal cancer cell lines and human specimens, we observed an inverse correlation between miR-101 and EP4 receptor protein. Transfection of LS174T cells with miR-101 significantly suppressed a luciferase reporter containing the EP4 receptor-3′-UTR. In contrast, a mutant EP4 receptor-3′-UTR was unaffected. Ectopic expression miR-101 markedly reduced cell proliferation and motility. Co-transfection of EP4 receptor could rescue colon cancer cells from the tumor suppressive effects of miR-101. Moreover, pharmacologic inhibition of EP4 receptor signaling or silencing of EP4 receptor phenocopied the effect of miR-101. This is the first study to show that the EP4 receptor is negatively regulated by miR-101.

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 2300. doi:1538-7445.AM2012-2300
Abstract 2304: MicroRNA-7 functions as antimetastic microRNA in gastric cancer by targeting insulin-like growth factor 1 receptor
Authors: Zhao Xiaodi , Dou Weijia , Nie Yongzhan , Fan Daiming
Published: August 2012

Metastasis is a major clinical obstacle in the treatment of gastric cancer (GC) and accounts for the majority of cancer-related mortality. MicroRNAs (miRNAs) have been recently emerged as regulators of metastasis through acting on multiple signaling pathways. In this study, we found that miR-7 is significantly down-regulated in high-metastatic GC cell lines and metastatic tissues. Both gain-of-function and loss-of-function studies showed that increased miR-7 expression significantly reduced migration and invasion of GC cells, while decreased miR-7 expression dramatically enhanced cell migration and invasion. In vivo metastasis assays also shown that overexpression of miR-7 remarkably inhibits GC metastasis. Moreover, the IGF1R oncogene, which is often mutated or amplified in human cancers and has been shown to function as an important regulator of cell growth and tumor invasion, was identified as a direct target of miR-7. RNA interference silencing of IGF1R recapitulated the antimetastic function of miR-7, whereas restoration of IGF1R expression attenuated the function of miR-7 in GC cells. Furthermore, we found that suppression of Snail by miR-7 through targeting IGF1R increased E-cadherin expression and partly reversed the epithelial-mesenchymal transition (EMT). Finally, analyses of the miR-7 and IGF1R level in human primary GC with matched lymph node metastasis tissue arrays revealed that miR-7 is inversely related to the increase of IGF1R expression. The present study provides an insight into the specific biological behavior of miR-7 in EMT and tumor metastasis. This novel miR-7/IGF1R/Snail axis would be helpful in the therapeutic application to block GC metastasis.

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 2304. doi:1538-7445.AM2012-2304
Abstract 2306: Overexpression of microRNA-223 regulates the ubiquitin ligase FBXW7 in esophageal squamous cell carcinoma
Authors: Kurashige Junji , Watanabe Masayuki , Iwatsuki Masaaki , Baba Yoshifumi , Baba Hideo
Published: August 2012

Background: F-box and WD repeat domain-containing 7 (FBXW7) is a cell cycle regulatory gene whose protein product ubiquitinates positive cell cycle regulators such as c-Myc, cyclin E and c-Jun, thereby acting as a tumor-suppressor gene. This study focused on microRNA-223 (miR-223), which is a candidate regulator of FBXW7 mRNA. The aim of this study was to clarify the clinical significance of miR-223 and FBXW7 in esophageal squamous cell carcinoma (ESCC) patients and to elucidate the mechanism by which FBXW7 is regulated by miR-223. Methods: The expression levels of miR-223 and the expression of FBXW7 protein was examined using 109 resected specimens to determine the clinicopathological significance. The expression levels of miR-223 were determined by qRT-PCR. The immunohistochemical studies for FBXW7 were performed on formalin-fixed paraffin-embedded surgical sections obtained from 109 patients with ESCC. We also investigated the role of miR-223 in the regulation of FBXW7 expression in ESCC cell lines in an in vitro analysis. Results: We found that the mean expression levels of miR-223 in cancerous tissue specimens were significantly higher than those in non-cancerous tissues (p <0.001). We divided the 109 ESCC patients into two groups according to the ratio of their cancer/normal tissue expression levels of miR-223, as α1.0 or <1.0 for the cancer/noncancerous tissues expression levels of miR-223. There were 74 cases (67.9%) in the high miR-223 group and 35 cases (32.1%) in the low miR-223 expression group. There were significant differences in gender (p = 0.008), tumor size (p = 0.042), and depth of tumor invasion (p = 0.030) between the groups. There was a significant inverse relationship between the expression levels of miR-223 and FBXW7 protein. High levels of miR-223 were associated with low FBXW7 expression (Pearson correlation, r = −0.336; p<0.01). Moreover, patients with high miR-223 expression demonstrated a significantly poorer prognosis than those with low expression (p=0.034). Based on a series of gain-of-function and loss-of-function investigation in vitro, we found that these effects may be due to the downregulation of the tumor-suppressor FBXW7, which was targeted by miR-223. Conclusions: Our present study indicates that high expression of miR-223 had a significant adverse impact on the survival of esophageal squamous cell carcinoma patients through repression of the function of FBXW7.

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 2306. doi:1538-7445.AM2012-2306
Abstract 231: Development of an ELISA for quantification of in vitro and cellular PTEN phosphatase activity
Authors: Landward Michael B. , Ehrmantrout Kimberly K. , Morrow Xin Chen , Branch Angie M. , Ferguson Colin G.
Published: August 2012

Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) is a 3′ phosphoinositide phosphatase that converts PI(3,4,5)P3 to PI(4,5)P2 thus opposing PKB/Akt activation by PI 3-Kinase. PTEN is involved in neuronal stem cell proliferation and self-renewal, cardiac myocyte hypertrophy and contractility, and a wide range of developmental processes. However, PTEN is best known for its role as a tumor suppressor. Loss of PTEN activity results in accumulation of PI(3,4,5)P3, abnormal activation of PKB/Akt, unregulated cell growth, suppression of apoptosis, and increased tumorigenesis in a number of human tissues. It has also been proposed that PTEN is a candidate for targeted chemotherapy because certain anti-cancer agents preferentially destroy tumors with PTEN mutations. In addition to this direct role in cancer, PTEN has recently been shown to regulate cancer-associated pathways including VEGF-mediated angiogenesis and others. Consequently interest in PTEN as a cancer target is still high. Traditionally, quantification of PTEN phosphatase activity has required the use of radioactivity, organic solvents, and chromatography. In addition, detection of free phosphate released from the PTEN activity is commonly used for quantification. However, this latter method has limited sensitivity and dynamic range, and is susceptible to contamination from other sources of inorganic phosphate which can give incorrect results. To address the need for a better quantification method, we have developed a PTEN activity assay using a standard competitive ELISA format. Rather than detecting free phosphate, the assay detects the PTEN product, PI(4,5)P2, giving much more accurate quantification, improved sensitivity, and eliminates possible errors from phosphate contamination. We have shown that the assay can successfully quantify PTEN activity using both recombinant and immunoprecipitated enzyme. Several cell lines were tested for PTEN activity including the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 (PTEN deficient).

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 231. doi:1538-7445.AM2012-231
Abstract 2310: Identification of signaling pathways involved in the regulation of let-7 in ovarian cancer
Authors: Guo Rong , Morin Patrice
Published: August 2012

miRNAs represent a class of small non-coding RNAs that can regulate the stability and translation of specific mRNA targets. Several members of the let-7 miRNA family are down-regulated in ovarian and other cancers and are thought to act as tumor suppressors, possibly through their ability to target growth-promoting genes (RAS, HMGA2), cell cycle genes (CDK6, CDK7, STK6, STK12), and anti-apoptotic genes (Bcl-2, casp3). However, the exact mechanisms regulating their expression and activity are not clearly known. In order to address this issue, we have developed a reporter assay for let-7 activity. We have made several reporter constructs containing the luciferase gene attached to different site combinations and sequences of let-7 in its 3′ UTR, and have identified a reporter that is extremely sensitive to let-7 activity. Ovarian cancer cell lines BG1 and UCI101 were transfected with the let-7 reporter and treated with 80 different kinase inhibitors in order to identify upstream pathways that can affect let-7 activity. We found nine inhibitors which caused consistent, dose-dependent changes on endogenous let-7 activity. The nine drugs can be categorized into three classes of inhibitors and further validation is currently underway through knockdowns of specific kinases. Our findings suggest novel molecular pathways involved in the regulation of let-7 family and, considering the important role of let-7 family members in ovarian cancer, these pathways may further our understanding of ovarian tumorigenesis and provide new targets for therapy.

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 2310. doi:1538-7445.AM2012-2310
Abstract 2316: Itraconazole, an antifungal drug with anti-angiogenic activity, inhibits VEGFR2 trafficking, glycosylation, and signaling in endothelial cells
Authors: Nacev Benjamin A. , Grassi Paola , Dell Anne , Haslam Stuart M. , Liu Jun O.
Published: August 2012

Itraconazole is a widely used antifungal drug that has been recently shown to inhibit angiogenesis in vivo and to block non-small cell lung cancer growth in animal models. Based on these and other results, itraconazole is currently being evaluated in four clinical trials as a cancer therapeutic. Angiogenesis, one of the hallmarks of cancer, is required for tumor growth and is driven by tumor-secreted growth factors including vascular endothelial growth factor (VEGF). We now report that in endothelial cells, itraconazole inhibits signaling of VEGF through the VEGF receptor 2 (VEGFR2) (80% inhibition; p < 0.05) and subsequently through a downstream substrate, PLCϒ1 (60% inhibition; p < 0.05). Mechanistically, we show that this was due to a nearly complete loss of VEGF binding to VEGR2 that was secondary to a loss of cell surface expression of the receptor. After itraconazole treatment, VEGFR2 was retained in a perinuclear aggregate which partially overlapped with the cis-Golgi marker GM130. Correlated with this loss of VEGFR2 signaling and trafficking, was a dramatic change in VEGFR2 mobility on SDS-PAGE which resulted from incomplete N-glycosylation. Itraconazole globally reduced poly-LacNAc and tetra-antennary glycans in endothelial cells and induced hypoglycosylation of EGFR in a renal cell carcinoma line, suggesting that the effects of itraconazole apply to multiple cell types and receptors. Importantly, small molecule inducers of lysosomal accumulation and mTOR inhibition, two previously known itraconazole-induced effects, did not interfere with VEGFR2 glycosylation. Similarly, glycosylation inhibitors did not affect cholesterol trafficking or inhibit mTOR. However, increasing cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, restored VEGFR2 glycosylation and signaling. Thus, the newly described properties of itraconazole occur in parallel to inhibition of cholesterol trafficking and mTOR but appear downstream of a single target. Taking these findings together, we propose a model in which itraconazole inhibits intracellular trafficking. This model could explain the loss of VEGFR2 signaling, incomplete VEGFR2 glycosylation, as well as disruption of cholesterol trafficking and mTOR signaling. Importantly, the effects of itraconazole we report here may significantly contribute to the in vivo efficacy of itraconazole and are relevant to ongoing 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 2316. doi:1538-7445.AM2012-2316
Abstract 2318: Azadirachta indica leaf extract attenuates angiogenesis in endothelial cells
Authors: Mahapatra Saswati , Young Charles Y.F , Tindall Donald J. , Donkena Krishna Vanaja
Published: August 2012

Azadirachta indica (common name: neem) leaves have been found to possess immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Neem leaves contain multiple bioactive compounds that could target multiple signaling pathways dysregulated in cancer. The present study evaluates anti-angiogenic potential of ethanol extract of neem leaves (EENL) in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with EENL inhibited VEGF induced angiogenic response, assessed by the formation of tubes in vitro and the formation of vascular structure in the angioreactor in vivo. The in vitro proliferation, invasion and migration of HUVECs were suppressed with EENL. Nuclear fragmentation and abnormally small mitochondria with dilated cristae were observed in EENL treated HUVECs by transmission electron microscopy. Genome-wide mRNA expression profiling after treatment with EENL revealed differentially regulated genes. Expression changes for the 30 up-regulated and 30 down-regulated genes were validated by quantitative real-time polymerase chain reaction. Additionally, increase in the expression of HMOX1, ATF3 and EGR1 proteins were determined by immunoblotting. Analysis of the compounds in the EENL by mass spectrometry with mobile phase containing sodium acetate suggests the presence of nimbolide, 2′, 3′-dehydrosalannol, 6-desacetyl nimbinene and nimolinone. We further confirmed antiproliferative activity of nimbolide and 2′, 3′-dehydrosalannol in HUVECs. Our results suggest that EENL by regulating the genes involved in cellular development and cell death functions could control cell proliferation, attenuate the stimulatory effects of VEGF and exert antiangiogenic effects. EENL treatment could have a potential therapeutic role during cancer progression.

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 2318. doi:1538-7445.AM2012-2318
Abstract 2323: Iron chelator contributes to anti-angiogenic therapy via selective induction of VEGF-A
Authors: Ohara Toshiaki , Noma Kazuhiro , Nishitani Seishi , Watanabe Shinichiro , Tomono Yasuko , Hashimoto Yuuri , Tazawa Hiroshi , Fujiwara Toshiyoshi
Published: August 2012

[Introduction] Iron is essential for cancer to survive. Cancer cell is suppressed to proliferate under the iron depletion condition using iron chelator. However, only iron depletion can't suppress tumor growth completely. The reason is that cancer cell has abilities to escape the severe environment. These abilities are known as angiogenesis, migration, etc. Thus, to inhibit not only cancer cell proliferation but also these abilities are important for anti-cancer therapy. Bevacizumab, an antibody against vascular endothelial growth factor, is the first clinical anti-angiogenic drug. It's used for many kinds of cancer patients and revealed clinical benefit. According to spread widely in use, the resistance of Bevacizumab has been recognized. Recently, patients in resistance of Bevacizumab were reported to be high concentration of PIGF, VEGF-C and VEGF-D. On the other hand, we have demonstrated a synergistic therapeutic effect of iron depletion on anti-angiogenic therapy for human cancer in this meeting. In vitro study, iron chelator was used to simulate the iron depletion condition. Our result showed that iron depletion has anti-proliferative effect and induction of VEGF-A. Therefore, Bevacizumab revealed a synergistic therapeutic effect in iron depletion condition. In this study, we hypothesized that iron chelation therapy has anti-proliferative effect and induction of only VEGF-A(not PIGF, VEGF-C and VEGF-D). [Matherials and Methods] A549, H1299 NSCLC cell lines were used. Iron chelator deferasirox and deferoxamine were prepared to simulate iron depletion condition. Cell viability was determined by WST-1 assay. To determine induction of iron chelator, PIGF, VEGF-A, C, D ELISA assays were performed. In vivo Bevacizumab administration study, we used A549 subcutaneous tumor xenograft mice(n=5/group). Deferasirox(50mg/kg) was administrated orally 5days in a week. Bevaicuzmab(5 mg/kg) was administrated twice a week intravenously. [Results] Iron chelator suppressed cancer cell proliferation and induced only VEGF-A(not PIGF, VEGF-C and VEGF-D). VEGF-A was induced by iron chelator in dose dependent manner. Deferasirox revealed synergistic anti-tumor effect with Bevacizumab in vivo study. [Conclusion] Iron chelator contributes to anti-angiogenic therapy via selectively inducing VEGF-A. Iron chelator can be a new therapeutic strategy of Bevacizumab combination therapy.

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 2323. doi:1538-7445.AM2012-2323
Abstract 2325: Increase in neuroblastoma metastasis after dual inhibition of VEGF and Notch
Authors: Garcia Alejandro , Banerjee Debarshi , Kitajewski Jan K. , Kandel Jessica J. , Yamashiro Darrell J.
Published: August 2012

Purpose: Vascular endothelial growth factor (VEGF) inhibition is a validated cancer treatment. However, even in responsive tumors, acquired resistance is common. Notch proteins also function as key angiogenic effectors, and cross-regulate VEGF expression, raising the question of whether combined treatment would enhance tumor suppression. We hypothesized that dual VEGF/Notch blockade would inhibit tumor growth in experimental SY5Y neuroblastoma. Methods: SY5Y neuroblastoma cells were lentivirally transfected to express Notch1-decoy (N1D) or GFP (control). Proliferation was assessed in vitro under both hypoxia and normoxia using BrdU assays. To examine the effect of N1D on tumor growth, 10[6] cells were xenografted intrarenally in nude mice and treated with placebo or bevacizumab (BV) twice a week. Tumor progression was monitored by bioluminescence. Metastatic burden in target organs was quantified by bioluminescence and histology. Vascular disruption due to N1D and BV treatment was evaluated by immunostaining. IACUC approval was obtained for all experiments. Results: Expression of soluble N1D was confirmed by immunoblotting conditioned media. SY5Y+N1D cells proliferated more rapidly in hypoxia than control SY5Y+GFP cells (p<0.01). In vivo, BV treatment alone significantly reduced tumor growth as compared to placebo (p=0.026). However, combined treatment (N1D + BV) did not further reduce SY5Y tumor growth as compared to BV treatment alone (p=0.684). Interestingly, dual-treated mice developed higher metastatic burdens in liver than mice treated with either agent alone or controls by bioluminescence (p=0.006). By histology, dual-treated mice displayed a higher incidence of liver metastasis (7/9 mice) vs. mice treated with BV only (3/10 mice). Immunostaining demonstrated disruption of tumor vessel architecture in BV- and N1D + BV-treated mice. Conclusion: Dual VEGF/Notch targeting of SY5Y tumors resulted in increased metastatic burden, without affecting primary tumor growth, as compared to either treatment alone. Recent data indicates that tumoral hypoperfusion can promote progression, potentially by selecting for biologically aggressive behaviors. Our results suggest that dual Notch/VEGF blockade causes an enhanced propensity to metastasize. These data warrant further preclinical investigation.

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 2325. doi:1538-7445.AM2012-2325
Abstract 2326: Antiangiogenic effects associated with the inhibition of HSP90 in colorectal cancer
Authors: Mahaseth Hemchandra , Nagaraju Ganji Purnachandra , Diaz Roberto , Taliaferro-Smith LaTonia D. , Landry Jerome C. , Saba Nabil F. , El-Rayes Bassel F.
Published: August 2012

Purpose/Objectives: Angiogenesis has a central role in tumor growth and metastasis. Inhibition of angiogenesis has been shown to improve outcomes of patients with advanced stage colorectal cancer receiving standard chemotherapy regimens. HSP90 has a central role in the regulation of transcription factors that modulate the expression of angiogenic cytokines. Ganetespib is a small molecule functional inhibitor of HSP90. We tested the hypothesis that HSP90 inhibition by ganetespib can inhibit angiogenesis in colorectal cell lines. Methods: Two colorectal cancer (CRC) cell lines were used, HCT-116 and HT-29. Cells were either untreated (control) or treated with ganetespib at 50 nM for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of ganetespib on HIF-1alpha and vascular endothelial growth factor (VEGF) expression. ELISA for VEGF expression was performed on the medium of CRC cells to evaluate the effects of ganetespib on VEGF secretion. Tube formation assays and chicken egg chorioallantoic membrane (Egg CAM) assay were also performed. Results: Western blot analyses demonstrated significant (p<0.001) inhibition of HIF-1alpha and VEGF expression in both cell lines treated with ganetespib. RT-PCR demonstrated significant (p<0.0001) downregulation of VEGF transcription. Similarly, ELISA assay demonstrated significant inhibition of VEGF expression in the culture medium in both CRC cell lines. The HUVEC tube formation and the egg CAM assays further confirmed that treatment of the cell lines with ganetespib inhibited angiogenesis. Conclusions: HSP90 inhibition has significant antiangiogenic effects in preclinical models. Evaluating HSP90 inhibitors in conjugation with chemotherapy in colorectal cancer is a rational approach that could have a significant impact on the outcome of this deadly and common disease.

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 2326. doi:1538-7445.AM2012-2326