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787: Transferrin-conjugated self assembled nanoparticles incorporating ZOL as a tool for the targeting of glioblastoma
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 784 A strategy to screen and subsequently identify therapeutically valuable microRNAs that target a clinically established KITENIN oncogene in colorectal cancer S.M. Yoon1 , S.Y. Park2 , J.A. Bae1 , Y.S. Ko1 , H.G. Kim2 , K.K. Kim1 . 1 Chonnam National University Medical School, Pharmacology, Gwangju, South Korea, 2 Sunchon National University, Pharmacology, Sunchon, South Korea Background and Method: Deregulation of microRNAs (miRNAs, miRs) in colorectal cancer (CRC) can contribute to cancer development and altered expression of miRNAs is causally associated with the initiation and progression of CRC. However, the regulation of CRC cell motility by miRNAs and the consequent modulation of CRC progression are not fully understood. We previously observed that KITENIN (KAI1 C-terminal interacting tetraspanin) is highly expressed in sporadic human CRC tissues and that the functional KITENIN complex acts to promote progression of CRC. However, it remains unknown whether KITENIN-targeting microRNAs modulate CRC cell motility and colorectal tumorigenesis. We here developed a strategy to initially screen for miRNAs that effectively target a clinically established oncogene in colorectal tumorigenesis, such as KITENIN and to subsequently identify therapeutically valuable anti-oncomirs among them. Results: We identified KITENIN-targeting miRNAs by miRNA library screening and bioinformatic analyses, and selected three miRNAs, namely, miR-27a, miR-30b, and miR-124, for further analysis. These miRNAs suppressed the invasion of several CRC cell lines, as shown by functional studies with synthetic miRNA precursors and inhibitors. We also screened for a final candidate via conditional expression of mature miRNAs in a mouse xenograft tumor model using a tetracycline-inducible system, and selected miR-124 as a potential anti-oncomir. Using a constitutive precursor miRNA overexpression system, which ensures biologically relevant interactions with regulatory partners, we confirmed that miR-124 functions as an effective suppressor of colorectal tumorigenesis. To identify other targets of miR-124, we subsequently performed mRNA microarray analysis on isolated tumor tissues from a xenograft tumor model in which precursor miR-124 was overexpressed and found that expression levels of MYH9 and SOX9 were significantly lower in the pre-miR-124-delivered tumor tissues. We also found that MYH9 and SOX9 are real targets of miR-124 in CRC cells using expression analyses. Conclusions: Our results demonstrated that miR-124 suppresses in vivo colorectal tumorigenesis by targeting MYH9 and SOX9 as well as KITENIN. We suggest that miR-124 among the KITENIN-targeting microRNAs is a therapeutically valuable anti-oncomir for CRC. No conflict of interest. 785 Multicellular tumor spheroid 3D models to decipher cancer cell biology and to evaluate anticancer drugs V. Lobjois1 , B. Ducommun2 . 1 ITAV − CNRS USR3505, Toulouse, France, 2 ITAV − CNRS USR3505, CNRS, Toulouse, France Introduction: Models that closely mimic human cancer are essential for the understanding of the intimate growth mechanisms and for the development of new treatments. MultiCellular Tumor Spheroid (MCTS) is a 3D model that accurately reproduces the organization of a microtumor, recapitulating cell– cell, cell-microenvironment and cell heterogeneity similar to what is found in microregions of a tumor. MCTS is therefore considered as an attractive model to evaluate the activity of new antiproliferative drugs. To fully exploit the features of MCTS, instead of a global assessment, it is essential to preserve the 3D regionalization level of analysis when investigating the effect of a drug. Material and Method: We report here the development of original MCTS expressing fluorescent reporters and markers that can be used to explore the regionalization and the dynamics of the effects of anticancer drugs in 3D using light sheet microscopy (SPIM). Results and Discussion: We will demonstrate the potential of these 3D models to study the kinetics aspects of the response on live spheroids. We will also report the development of innovative microdevices dedicated to the study of the dynamics of MCTS formation and to the monitoring of mechanosensing and tumor growth-induced mechanical stress. Conclusion: We will present our latest advances on the characterization of these processes and on the use of these assays for the identification of new original targets and compounds. No conflict of interest. 786 The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma E. Rovida1 , G. Di Maira2 , S. Cannito3 , I. Tusa1 , C. Paternostro3 , X. Deng4 , P. Dello Sbarba1 , N.S. Gray4 , M. Parola3 , F. Marra2 . 1 University of Firenze, DSBSC, Firenze, Italy, 2 University of Firenze, Dipartimento di Medicina Sperimentale e Clinica, Firenze, Italy, 3 University of Torino, Dipartimento di Medicina e Oncologia Sperimentali, Firenze, Italy, 4 Harvard Medical School, Dana-Farber Cancer Institute, Boston, USA Background: The Extracellular Signal-Regulated kinase 5 (ERK5 or BMK1) is involved in tumour development. The ERK5 gene may be amplified in
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hepatocellular carcinoma (HCC) but its biological role has not been clarified. In this study we explored the role of ERK5 expression and activity in HCC in vitro and in vivo. Material and Methods: ERK5 expression was evaluated in human liver tissue. Cultured HepG2 and Huh-7 were studied after ERK5 knockdown by siRNA or in the presence of the specific pharmacologic inhibitor, XMD8−92. The role of ERK5 in vivo was assessed using mouse Huh-7 xenografts. Results: In tissue specimens from patients with HCC, a higher percentage of cells with nuclear ERK5 expression was found both in HCC and in the surrounding cirrhotic tissue, compared to normal liver tissue. Treatment of Huh-7 or HepG2 cells with XMD8−92 or with ERK5 siRNA decreased HCC cell proliferation, increased the proportion of cells in G0/G1 phase, and augmented the expression of p27Kip. Genetic or pharmacologic inhibition of ERK5 prevented cell migration induced by EGF or hypoxia, and caused cytoskeletal remodelling and rearrangement of focal adhesions. In mouse xenografts, the rate of tumour appearance and the size of tumours were significantly lower when Huh-7 were silenced for ERK5. Moreover, systemic treatment with XMD8−92 of mice with established HCC xenografts markedly reduced tumour growth and decreased the expression of the proto-oncogene c-Rel. Conclusions: ERK5 regulates the biology of HCC cells and modulates tumour development and growth in vivo. This pathway should be investigated as a possible therapeutic target in HCC. No conflict of interest. 787 Transferrin-conjugated self assembled nanoparticles incorporating ZOL as a tool for the targeting of glioblastoma M. Caraglia1 , S. Zappavigna1 , M. Porru2 , L. Amalia1 , S. Lusa3 , G. Salzano3 , S. Artuso2 , A. Stoppacciaro2 , C. Leonetti2 , G. De Rosa4 . 1 Second University of Naples, Department of Biochemistry Biophysics and General Pathology, Naples, Italy, 2 Regina Elena Cancer Institute, Department of Experimental Oncology, Rome, Italy, 3 University “Federico II”, Department of Pharmacy, Naples, Italy, 4 University “Federico II”, Department of Pharmacy, Rome, Italy Background: Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. The blood–brain barrier (BBB) is the most important limiting factor for the development of new drugs and drug delivery for the central nervous system (CNS). Different methods have been used to facilitate the drug transportation into the brain. Here, we propose a new strategy to treat glioblastoma based on self-assembling nanoparticles (NPs) encapsulating Zoledronic Acid (ZOL). Material and Methods: NPs are obtained by spontaneously assembling of ZOL, inorganic NPs and PEGylated liposomes (NPs-ZOL). In order to improve the targeting of the brain tumor, these NPs have been functionalized with transferrin (Tf), maintaining the possibility to prepare them before use by selfassembling procedure (NPs-ZOL-Tf). The in vitro cytotoxic activity of NPs were evaluated on different glioblastoma cell lines by MTT assay. In vivo experiments were performed on glioblastoma xenografts to compare the antitumor efficacy of NPs-ZOL to free ZOL, in terms of percent tumor weight inhibition (TWI%), tumor growth delay (T-C) and percent increase of mice survival (ILS%). Results: The encapsulation in NPs resulted in higher in vitro cytotoxic activity than free ZOL on LN229, U373MG and U87MG glioblastoma cells. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was equal (LN229) or less (U373MG and U87MG) than that one induced by NPsZOL not functionalized with Tf and correlated with Tf receptor expression on tumour cells. On the other hand, Tf-NPs-ZOL showed a higher antitumor efficacy if compared with that one caused by naked NPs-ZOL in mice-bearing glioblastoma tumors, inducing a significant TWI of 41%, a T-C of 10 days and an increase of ILS of 23%. Interestingly, while NPs-ZOL treatment induced a stable disease in 1 out of six mice treated, NPs-ZOL-Tf produced a complete tumor regression in 1 out of six mice treated. The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the good efficacy of NPs-ZOL-Tf. Indeed, NPs-ZOL administration elicited the stabilization of the disease in 2 out of eight mice treated and an overall increase of survival of 13%, NPs-ZOL-Tf treatment resulted in an improved therapeutic efficacy as 3 out of eight mice showed a stable disease and, impressively, a complete tumor response was reported in one mouse. Finally, an overall increase of survival of 23% was observed. These effects were paralleled by an higher uptake and intratumour localization of fluorescently-labeled-NPs-Tf in LN229 cells and both in intramuscularly and intracranial xenografts. Discussion: Our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB, thus suggesting the application of this strategy in clinical setting. No conflict of interest.
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hepatocellular carcinoma (HCC) but its biological role has not been clarified. In this study we explored the role of ERK5 expression and activity in HCC in vitro and in vivo. Material and Methods: ERK5 expression was evaluated in human liver tissue. Cultured HepG2 and Huh-7 were studied after ERK5 knockdown by siRNA or in the presence of the specific pharmacologic inhibitor, XMD8−92. The role of ERK5 in vivo was assessed using mouse Huh-7 xenografts. Results: In tissue specimens from patients with HCC, a higher percentage of cells with nuclear ERK5 expression was found both in HCC and in the surrounding cirrhotic tissue, compared to normal liver tissue. Treatment of Huh-7 or HepG2 cells with XMD8−92 or with ERK5 siRNA decreased HCC cell proliferation, increased the proportion of cells in G0/G1 phase, and augmented the expression of p27Kip. Genetic or pharmacologic inhibition of ERK5 prevented cell migration induced by EGF or hypoxia, and caused cytoskeletal remodelling and rearrangement of focal adhesions. In mouse xenografts, the rate of tumour appearance and the size of tumours were significantly lower when Huh-7 were silenced for ERK5. Moreover, systemic treatment with XMD8−92 of mice with established HCC xenografts markedly reduced tumour growth and decreased the expression of the proto-oncogene c-Rel. Conclusions: ERK5 regulates the biology of HCC cells and modulates tumour development and growth in vivo. This pathway should be investigated as a possible therapeutic target in HCC. No conflict of interest. 787 Transferrin-conjugated self assembled nanoparticles incorporating ZOL as a tool for the targeting of glioblastoma M. Caraglia1 , S. Zappavigna1 , M. Porru2 , L. Amalia1 , S. Lusa3 , G. Salzano3 , S. Artuso2 , A. Stoppacciaro2 , C. Leonetti2 , G. De Rosa4 . 1 Second University of Naples, Department of Biochemistry Biophysics and General Pathology, Naples, Italy, 2 Regina Elena Cancer Institute, Department of Experimental Oncology, Rome, Italy, 3 University “Federico II”, Department of Pharmacy, Naples, Italy, 4 University “Federico II”, Department of Pharmacy, Rome, Italy Background: Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. The blood–brain barrier (BBB) is the most important limiting factor for the development of new drugs and drug delivery for the central nervous system (CNS). Different methods have been used to facilitate the drug transportation into the brain. Here, we propose a new strategy to treat glioblastoma based on self-assembling nanoparticles (NPs) encapsulating Zoledronic Acid (ZOL). Material and Methods: NPs are obtained by spontaneously assembling of ZOL, inorganic NPs and PEGylated liposomes (NPs-ZOL). In order to improve the targeting of the brain tumor, these NPs have been functionalized with transferrin (Tf), maintaining the possibility to prepare them before use by selfassembling procedure (NPs-ZOL-Tf). The in vitro cytotoxic activity of NPs were evaluated on different glioblastoma cell lines by MTT assay. In vivo experiments were performed on glioblastoma xenografts to compare the antitumor efficacy of NPs-ZOL to free ZOL, in terms of percent tumor weight inhibition (TWI%), tumor growth delay (T-C) and percent increase of mice survival (ILS%). Results: The encapsulation in NPs resulted in higher in vitro cytotoxic activity than free ZOL on LN229, U373MG and U87MG glioblastoma cells. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was equal (LN229) or less (U373MG and U87MG) than that one induced by NPsZOL not functionalized with Tf and correlated with Tf receptor expression on tumour cells. On the other hand, Tf-NPs-ZOL showed a higher antitumor efficacy if compared with that one caused by naked NPs-ZOL in mice-bearing glioblastoma tumors, inducing a significant TWI of 41%, a T-C of 10 days and an increase of ILS of 23%. Interestingly, while NPs-ZOL treatment induced a stable disease in 1 out of six mice treated, NPs-ZOL-Tf produced a complete tumor regression in 1 out of six mice treated. The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the good efficacy of NPs-ZOL-Tf. Indeed, NPs-ZOL administration elicited the stabilization of the disease in 2 out of eight mice treated and an overall increase of survival of 13%, NPs-ZOL-Tf treatment resulted in an improved therapeutic efficacy as 3 out of eight mice showed a stable disease and, impressively, a complete tumor response was reported in one mouse. Finally, an overall increase of survival of 23% was observed. These effects were paralleled by an higher uptake and intratumour localization of fluorescently-labeled-NPs-Tf in LN229 cells and both in intramuscularly and intracranial xenografts. Discussion: Our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB, thus suggesting the application of this strategy in clinical setting. No conflict of interest.