Au cours de cette thèse, j’ai utilisé des complexes composés d’acides nucléiques, d’un polymère cationique et de liposomes cationiques appelés Lipopolyplexes pour formuler des siRNA (LPRi) et un leurre ADN (LPD) afin inhiber la croissance des cellules 4T1, un modèle murin de carcinome mammaire. Dans une première étude, des injections systémique ou endotrachéale de LPRi avec des siRNA anti-luciférase n’ont pas permis d’inhiber l’expression de la luciférase dans des métastases pulmonaires induites par des cellules 4T1-luciférase. A partir de ces résultats, les LPRi ont été améliorés en ciblant les cellules 4T1 avec le peptide uPA et/ou RGDc ou l’acide folique incorporés aux liposomes selon diverses approches. Les formulations obtenues ont été caractérisées, leur endocytose et l’effet siRNA mesurés in vitro. Cette deuxième partie a permis d’établir que les LPRi décorés avec du folate étaient la meilleure formulation ciblée. Dans une troisième partie, l’inhibition de la prolifération des cellules 4T1 a été recherchée en ciblant le facteur de transcription STAT3. Des LPRi anti-STAT3 ont montré une très bonne efficacité pour inhiber STAT3, mais sans effet antiprolifératif significatif. Des LPD anti-STAT3 ont montré un très bon effet antiprolifératif, celui-ci étant renforcé lorsqu’une co-délivrance siRNA/leurre ADN (LPRiD) a été réalisée. In vivo, un délai de la croissance des tumeurs 4T1 a été observé après co-délivrance siRNA/leurre ADN. Cette thèse a permis de montrer l’efficacité des lipopolyplexes pour la délivrance combinée de siRNA et de leurre ADN dans les cellules tumorales 4T1. Ils indiquent que des études sont cependant nécessaires pour augmenter leur délivrance in vivo dans la tumeur. / During this thesis, I used complexes made with nucleic acids, cationic polymer and cationic liposomes called Lipopolyplexes to formulate siRNA (LPRi) and DNA molecular decoy (LPD) in order to inhibit the growth of 4T1 cells, a murine model of mammary carcinoma. In a first study, systemic or endotracheal injections of LPRi comprising anti-luciferase siRNA did not allow luciferase inhibition in pulmonary metastases induced by 4T1-Luc cells. From these results, LPRi were improved by targeting 4T1 cells using incorporation, by different means, of uPA and/or RGDc peptide or folic acid in liposomes. Resulted formulations were characterized, their internalization and siRNA transfection efficiency were measured in vitro. This second part showed that folate targeting of LPRi was the best formulation. In a third part, proliferation inhibition of 4T1 cells was investigated by targeting the STAT3 transcription factor. Anti-STAT3 siRNA LPRi showed very good efficacy in inhibiting STAT3, but without significant antiproliferative effect. Anti-STAT3 decoy LPD showed a very good antiproliferative effect, the latter being reinforced when co-delivery siRNA/DNA decoy (LPRiD) was performed. In vivo, a growth retardation of 4T1 tumors was observed after co-delivery siRNA/DNA decoy. This thesis demonstrated the effectiveness of lipopolyplexes for combined delivery of siRNA and DNA decoy in the 4T1 tumor cells. Some studies are however required to increase their in vivo delivery into the tumor.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24800号 / 医博第4992号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 貴浩, 教授 波多野 悦朗, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Increasingly the role of epigenetic machinery as a bridge between underlying DNA sequence and cellular phenotype is being discovered. The establishment of a myriad of unique cellular types sharing identical gene sequences in a multicellular organism gives a broad sense for the inherent role of epigenetic influence on cell differentiation. Importantly, the epigenetic mechanisms involved in establishing cell identity unsurprisingly contribute to diseased states, including cancer. Recent research continues to elucidate contributory roles of epigenetic mechanisms, such as DNA methylation, histone modification, and microRNA regulation, in human cancers. Additionally, chromatin remodelers, such as the Nucleosome Remodeling Factor (NURF), have been identified as important regulators for normal cell biology. While much has been done to identify and characterize the role of NURF chromatin remodeling complex as a key regulator of development in a number of model organisms, little has been published on the implications of NURF in diseases such as cancer. Our preliminary data shows dysregulation of E-cadherins, N-cadherins, and MHC-I genes in Bptf (an essential subunit of NURF) knocked down murine breast cancer cell lines. These proteins have well documented roles in the development and metastatic progression of cancers. To study the effect of Bptf knockdown on the development and progression of cancer we injected Bptf knocked down mouse breast cancer cell lines, 4T1, 66cl4, and 67NR, into syngenic BALB/c mice. Our findings reveal decreased tumor growth in 66cl4 and 67NR as measured by tumor weight at 3-4 weeks post injection. Tumor growth did not appear to be significantly affected in 4T1 challenged mice. However, mice inoculated with Bptf knockdown 4T1 cell lines have decreased metastasis to lungs as compared to control while metastasis of 66cl4 tumors to the lungs appear unaffected. To assess the role of the immune system in decreasing tumor growth in BALB/c mice, we injected 66cl4 tumors into NOD-SCID-Gamma (NSG) immune deficient mice. The tumors from these mice show no difference in tumor growth between Bptf knockdown and control tumors, implicating a role for the immune system regulating the decreased tumor weight in BALB/c mice. To delineate which immune cell effector may impede breast cancer carcinogenesis, we performed an in vitro natural killer (NK) cell cytotoxicity assay against 66cl4 tumors and found greater susceptibility to NK killing in Bptf knockdown tumors.

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