ASCT2 in Human Liver Cancer Cell Growth and Survival

ASCT2 在人肝癌细胞生长和存活中的作用

• 批准号: 6899478
• 负责人: BARRIE P BODE
• 金额: $ 22.05万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-18 至 2008-08-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6899478
• 关键词: aminoacid transport; antisense nucleic acid; apoptosis; biological signal transduction; cell line; cysteine endopeptidases; enzyme activity; flow cytometry; gene induction /repression; hepatocellular carcinoma; liver cells; microarray technology; neoplastic cell; oligonucleotides; phenotype; phosphatidylinositol 3 kinase; protein kinase; small interfering RNA; transfection

DESCRIPTION (provided by applicant): The broad, long-term objectives of this proposal are to evaluate the global potential of amino acid transporter ATB/ASCT2 as a selective therapeutic target in human hepatocellular carcinoma {HCC) and to elucidate the signaling mechanisms linking it to liver cancer cell growth and survival. Currently, there are no effective therapies for this cancer and its incidence continues to rise in the United States. ASCT2 is expressed in all human liver cancers examined to date, but is not expressed in normal human hepatocytes. Suppression of ASCT2 expression by inducible antisense RNA in the SK-Hep HCC cell line leads to cell death by the intrinsic apoptotic pathway via a mechanism that is attenuated by osmotic cell swelling. Therefore, the working hypothesis is that ASCT2, via its role in substrate delivery and cellular hydration (swelling) is linked to mammalian target of rapamycin (mTOR)- and phosphatidylinositol-3 kinase (PI3K)- dependent survival signaling pathways, respectively, that impinge upon proteins responsible for apoptotic suppression in all human liver cancer ceils. The aims of this proposal thus addresses the universality and mechanisms linking ASCT2 to hepatoma survival: Specific Aim 1: To evaluate the impact of ASCT2 post-transcriptional gene silencing (PTGS) on the growth and viability of six human hepatoma cell lines, representing a broad spectrum of differentiated phenotypes. Research Plan: Several individual candidate small interfering RNA (siRNA) specific to ASCT2 will be transfected into all six human hepatoma cell lines, and assessed for effects on growth, viability, ASCT2 expression and caspase activation, compared to appropriate control siRNA. Subsequently, adenoviral DMA vectors encoding for control and effective siRNA's (ideally 3) will be constructed and employed in signaling experiments. Off-target siRNA effects in ASCT2 silenced cells will be assessed by examination of a select panel of mRNA, microarray analysis and by the ability of reintroduced ASCT2 to "rescue" them. Alternative/corroborative approaches include use of PTGS methods such as morpholino antisense oligonucleotides or an inducible antisense RNA system. Specific Aim 2: To elucidate the signaling pathways linking ASCT2 silencing to hepatoma apoptosis. Research Plan: Signaling via PI3K-linked (PKB/Akt) and amino acid-dependent (mTOR) pathways as they relate to the pro-apoptotic Bcl-2 family member Bad, or other "mitochondrial gatekeepers", will be investigated pre- and post-ASCT2 silencing through the use of selective biochemical inhibitors and western blot analyses. Putative links established through this approach will be later confirmed by the expression of dominant negative or constitutively active variants of Akt or other signaling proteins. The impact of ASCT2 silencing on cell volume will be assessed by flow cytometry analysis, and reciprocally, the ability of artificial cell swelling and shrinkage to modulate these signaling pathways will also be investigated to better understand the mechanistic link between amino acid transporter function and cancer cell survival.

描述(由申请人提供)：这项建议的广泛、长期目标是评估氨基酸转运体ATB/ASCT2作为人类肝细胞癌(HCC)选择性治疗靶点的全球潜力，并阐明其与肝癌细胞生长和存活相关的信号机制。目前，这种癌症还没有有效的治疗方法，其发病率在美国继续上升。到目前为止，ASCT2在所有被检查的人肝癌中都有表达，但在正常人肝细胞中不表达。在SK-Hep肝癌细胞系中，可诱导的反义RNA抑制ASCT2的表达，通过渗透性细胞肿胀减弱的机制，通过内在的凋亡途径导致细胞死亡。因此，工作假说是，ASCT2通过其在底物输送和细胞水化(肿胀)中的作用，分别与哺乳动物雷帕霉素靶标(MTOR)和磷脂酰肌醇-3激酶(PI3K)依赖的生存信号通路相连，这些通路影响到所有人肝癌细胞中负责抑制凋亡的蛋白质。因此，本提案的目的在于探讨ASCT2与肝癌存活率之间的联系的普遍性和机制：具体目标1：评估ASCT2转录后基因沉默(PTGS)对代表多种分化表型的六种人肝癌细胞系生长和存活的影响。研究计划：将几个针对ASCT2的单个候选小干扰RNA(SiRNA)导入所有六个人肝癌细胞系，并评估其对生长、活力、ASCT2表达和 Caspase激活，与适当对照的siRNA相比。随后，编码控制和有效siRNA的腺病毒DMA载体(理想情况下为3)将被构建并用于信号转导实验。ASCT2沉默细胞中的非靶点siRNA效应将通过检测一组精选的信使核糖核酸、微阵列分析以及重新引入ASCT2来“拯救”它们的能力来评估。替代/佐证方法包括使用PTGS方法，如吗啉反义寡核苷酸或可诱导的反义RNA系统。特定目的2：阐明ASCT2沉默与肝癌细胞凋亡的信号转导途径。研究计划：通过PI3K连接(PKB/Akt)和氨基酸依赖(MTOR)通路与促凋亡的Bad或其他“线粒体守门人”相关的信号通路，将通过使用选择性生化抑制剂和蛋白质印迹分析，在ASCT2沉默前后进行研究。通过这种方法建立的假定的联系将在稍后被Akt或其他信号蛋白的显性负性或结构性活性变体的表达所证实。ASCT2沉默对细胞体积的影响将通过流式细胞仪分析来评估，反过来，人工细胞膨胀和收缩调节这些信号通路的能力也将被研究，以更好地了解氨基酸转运体功能和癌细胞存活之间的机制联系。