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）：该提案的广泛、长期目标是评估氨基酸转运蛋白 ATB/ASCT2 作为人类肝细胞癌 (HCC) 选择性治疗靶点的全球潜力，并阐明与其相关的信号传导机制影响肝癌细胞的生长和存活。目前，这种癌症还没有有效的治疗方法，并且其发病率在美国持续上升。 ASCT2在迄今为止检查的所有人类肝癌中表达，但在正常人肝细胞中不表达。在 SK-Hep HCC 细胞系中，诱导型反义 RNA 抑制 ASCT2 表达，通过渗透性细胞肿胀减弱的机制，通过内在凋亡途径导致细胞死亡。因此，工作假设是 ASCT2 通过其在底物输送和细胞水合（肿胀）中的作用，分别与哺乳动物雷帕霉素靶点 (mTOR) 和磷脂酰肌醇 3 激酶 (PI3K) 依赖性生存信号通路相关，这些信号通路分别影响负责所有人类肝癌细胞中细胞凋亡抑制的蛋白质。 因此，本提案的目的是解决 ASCT2 与肝癌存活相关的普遍性和机制： 具体目标 1：评估 ASCT2 转录后基因沉默 (PTGS) 对六种人肝癌细胞系的生长和活力的影响，这六种人肝癌细胞系代表了广泛的差异化表型谱。研究计划：将 ASCT2 特异性的几种候选小干扰 RNA (siRNA) 转染到所有六种人肝癌细胞系中，并评估对生长、活力、ASCT2 表达和 caspase 激活，与适当的对照 siRNA 相比。随后，将构建编码对照和有效 siRNA（最好是 3 个）的腺病毒 DMA 载体，并将其用于信号传导实验。 ASCT2沉默细胞中的脱靶siRNA效应将通过检查一组选定的mRNA、微阵列分析以及重新引入ASCT2“拯救”它们的能力来评估。替代/证实方法包括使用 PTGS 方法，例如吗啉代反义寡核苷酸或诱导型反义 RNA 系统。具体目标 2：阐明 ASCT2 沉默与肝癌细胞凋亡之间的信号通路。研究计划：通过 PI3K 相关 (PKB/Akt) 和氨基酸依赖性 (mTOR) 途径的信号传导，因为它们与促凋亡 Bcl-2 家族成员 Bad 或其他“线粒体看门人”相关，将在研究前和研究中进行研究通过使用选择性生化抑制剂和蛋白质印迹分析进行 ASCT2 后沉默。通过这种方法建立的推定联系稍后将通过 Akt 或其他信号蛋白的显性失活或组成型活性变体的表达得到证实。 ASCT2沉默对细胞体积的影响将通过流式细胞术分析进行评估，相应地，人工细胞肿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蛋白功能与癌细胞之间的机制联系生存。