Amino Acid Transporters ASCT2 and LAT1 in Human Hepatocellular Cancer Growth

氨基酸转运蛋白 ASCT2 和 LAT1 在人类肝细胞癌生长中的作用

• 批准号: 7516685
• 负责人: BARRIE P BODE
• 金额: $ 9.79万
• 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 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7516685
• 关键词: 3-Dimensional; Acids; Affect; Amino Acid Transporter; Amino Acids; Apoptosis; Biology; Biotinylation; Cancerous; Carrier Proteins; Cell Line; Cell membrane; Cells; Cellular Spheroids; Cessation of life; Chronic Hepatitis C; Complex; Confocal Microscopy; Cross-Linking Reagents; Cues; Depth; Disseminated Malignant Neoplasm; Epitopes; Goals; Growth; Growth and Development function; Heel; Hepatocyte; Human; Hypoxia; Immunoprecipitation; Incidence; Infection; Kinetics; Laboratories; Malignant Epithelial Cell; Malignant Neoplasms; Mediating; Modality; Modeling; Nutrient; Pathway interactions; Phenotype; Prevalence; Primary carcinoma of the liver cells; Process; Proteins; Public Health; RNA Interference; Radiolabeled; Regulation; Relative (related person); Research Project Grants; Role; Signal Pathway; Signal Transduction; Sirolimus; Small Interfering RNA; Stress; Surface; System; Testing; United States; Western Blotting; Work; base; cancer cell; clinically relevant; deprivation; extracellular; hepatoma cell; insight; outcome forecast; prospective; protein crosslink; radiotracer; resistance mechanism; response; small hairpin RNA; trafficking; tumor; uptake

DESCRIPTION (provided by applicant): This project aims to examine the efficacy of targeting amino acid transporters ASCT2 and LAT1 in human hepatocellular carcinoma (HCC), a recalcitrant cancer with poor prognosis and limited treatment options. ASCT2 and LAT1 are coordinately upregulated in a broad spectrum of primary human cancers, including HCC, but it is presently unclear what makes these two transporters (out of several) so coveted by malignancies. Neither transporter is expressed in normal liver cells (hepatocytes) at detectable levels making them attractive prospective targets for therapy. Moreover, ASCT2 and LAT1 have been shown to physically associate in the plasma membrane of human cancer cells, suggesting that they cooperate to help drive cancerous growth. Previous work from our laboratory showed that targeted silencing of ASCT2 elicits programmed cell death (apoptosis) in human HCC cells; here, we include LAT1 in the repertoire of transporter-targeted therapy. The primary objective of the currently proposed project is to test the broad applicability of targeted transporter therapy in HCC and in doing so, to assess the role of ASCT2 and LAT1 in HCC growth and survival signaling. This aim will be achieved through the use of 10 human hepatoma cell lines representing a broad clinically relevant spectrum of different HCC phenotypes. A stably maintained inducible expression system for short hairpin RNA (shRNA) targeting ASCT2 or LAT1 will be established in each of the 10 hepatoma lines, and utilized to assess the efficacy of targeting these transporters via RNA interference (RNAi) based silencing. The consequences of transporter silencing on growth and survival signaling (mTOR and Akt pathways, respectively) will be assessed through western blotting analysis. A second aim of the project will be to establish whether ASCT2 and LAT1 physically associate in the plasma membrane of HCC cells, and how silencing of one transporter affects the expression and activity of the other. The third aim of the project involves elucidating which cues present in the tumor microenvironment (amino acid limitation, hypoxia and acid pH) induce trafficking of the transporter complex to the plasma membrane where they function. In cases where transporter silencing does not induce apoptosis, their role in initial avascular growth (nascent tumor formation) will be assessed via spheroid formation (3-dimensional aggregation and growth of HCC cell lines). Collectively, these aims will give insights into the potential for developing transporter-targeted therapy, and the role of each in human HCC biology. PUBLIC HEALTH RELEVANCE: This project aims to investigate the potential of targeting two amino acid transporters upregulated in cancer ASCT2 and LAT1 as therapy for human hepatocellular carcinoma (HCC), and to determine their role in the development and growth of hepatocellular cancer. The incidence of HCC is on the rise in the United States due primarily to the increasing prevalence of chronic hepatitis C infection, and there are currently limited treatment options for this recalcitrant malignancy. Finding an "Achilles heel" in this cancer that is amenable to therapy is therefore paramount.

描述（由申请人提供）：该项目旨在研究靶向氨基酸转运蛋白 ASCT2 和 LAT1 在人肝细胞癌 (HCC) 中的功效，HCC 是一种预后不良且治疗选择有限的顽固性癌症。 ASCT2 和 LAT1 在包括 HCC 在内的多种原发性人类癌症中协同上调，但目前尚不清楚是什么让这两种转运蛋白（在多种转运蛋白中）如此受到恶性肿瘤的青睐。这两种转运蛋白在正常肝细胞（肝细胞）中都不以可检测的水平表达，这使得它们成为有吸引力的潜在治疗靶标。此外，ASCT2 和 LAT1 已被证明在人类癌细胞的质膜中存在物理关联，这表明它们合作帮助推动癌细胞的生长。我们实验室之前的工作表明，ASCT2 的靶向沉默会引发人类 HCC 细胞的程序性细胞死亡（细胞凋亡）；在这里，我们将 LAT1 纳入转运蛋白靶向治疗的范围内。目前提出的项目的主要目标是测试靶向转运蛋白治疗在 HCC 中的广泛适用性，并在此过程中评估 ASCT2 和 LAT1 在 HCC 生长和生存信号传导中的作用。这一目标将通过使用 10 种人类肝癌细胞系来实现，这些细胞系代表了不同 HCC 表型的广泛临床相关谱系。将在 10 个肝癌细胞系中建立针对 ASCT2 或 LAT1 的短发夹 RNA (shRNA) 的稳定维持的诱导表达系统，并用于评估通过基于 RNA 干扰 (RNAi) 的沉默靶向这些转运蛋白的功效。转运蛋白沉默对生长和生存信号（分别是 mTOR 和 Akt 途径）的影响将通过蛋白质印迹分析进行评估。该项目的第二个目标是确定 ASCT2 和 LAT1 是否在 HCC 细胞的质膜中物理结合，以及一种转运蛋白的沉默如何影响另一种转运蛋白的表达和活性。该项目的第三个目标是阐明肿瘤微环境中存在的哪些线索（氨基酸限制、缺氧和酸性 pH 值）诱导转运蛋白复合物转运至其发挥作用的质膜。在转运蛋白沉默不诱导细胞凋亡的情况下，它们在初始无血管生长（新生肿瘤形成）中的作用将通过球体形成（HCC 细胞系的 3 维聚集和生长）进行评估。总的来说，这些目标将深入了解开发转运蛋白靶向疗法的潜力，以及每种疗法在人类 HCC 生物学中的作用。公共健康相关性：该项目旨在研究针对癌症 ASCT2 和 LAT1 上调的两种氨基酸转运蛋白作为人类肝细胞癌 (HCC) 治疗的潜力，并确定它们在肝细胞癌发生和生长中的作用。在美国，肝癌的发病率呈上升趋势，主要是由于慢性丙型肝炎感染的患病率不断增加，而目前这种顽固性恶性肿瘤的治疗选择有限。因此，找到这种癌症中适合治疗的“致命弱点”至关重要。