Amino Acid Transporters ASCT2 and LAT1 in Human Hepatocellular Cancer Growth

描述(申请人提供)：该项目旨在研究针对氨基酸转运体ASCT2和LAT1在人类肝细胞癌(HCC)中的疗效。ASCT2和LAT1在包括肝癌在内的多种原发人类癌症中协同上调，但目前尚不清楚是什么原因使这两种转运蛋白如此受到恶性肿瘤的垂涎。这两种转运蛋白在正常肝细胞(肝细胞)中都不表达，这使它们成为有吸引力的潜在治疗靶点。此外，ASCT2和LAT1已被证明在人类癌细胞的质膜上物理上相关，这表明它们相互合作，帮助推动癌症的生长。我们实验室以前的工作表明，靶向沉默ASCT2会在人肝癌细胞中引起程序性细胞死亡(凋亡)；在这里，我们将LAT1包括在转运体靶向治疗的方程式中。目前提出的项目的主要目标是测试靶向转运蛋白治疗在肝癌中的广泛适用性，并在这样做的同时，评估ASCT2和LAT1在肝癌生长和生存信号中的作用。这一目标将通过使用10个人类肝癌细胞系来实现，这些细胞系代表了临床上广泛相关的不同肝癌表型的光谱。针对ASCT2或LAT1的短发夹状RNA(ShRNA)的稳定诱导表达系统将分别在10个肝癌细胞系中建立，并用于评估通过基于RNA干扰(RNAi)的沉默来靶向这些转运蛋白的效果。转运蛋白沉默对生长和生存信号(分别为mTOR和Akt通路)的影响将通过蛋白质印迹分析来评估。该项目的第二个目标将是确定ASCT2和LAT1是否在肝癌细胞的质膜上存在物理联系，以及沉默一个转运体如何影响另一个转运体的表达和活性。该项目的第三个目标涉及阐明肿瘤微环境中存在的哪些信号(氨基酸限制、低氧和酸性pH)导致转运体复合体运输到它们发挥作用的质膜。在转运体沉默不能诱导细胞凋亡的情况下，它们在初始无血管生长(新生肿瘤的形成)中的作用将通过球体形成(肝细胞癌细胞系的三维聚集和生长)来评估。总而言之，这些目标将使人们深入了解开发转运蛋白靶向治疗的潜力，以及每种治疗方法在人类肝癌生物学中的作用。公共卫生相关性：该项目旨在研究靶向ASCT2和LAT1中上调的两种氨基酸转运蛋白作为治疗人类肝细胞癌(HCC)的可能性，并确定它们在肝细胞癌的发生和生长中的作用。在美国，肝癌的发病率正在上升，这主要是由于慢性丙型肝炎感染的发病率增加，而目前对这种顽固性恶性肿瘤的治疗选择有限。因此，在这种癌症中找到一个可以接受治疗的“阿喀琉斯之踵”是至关重要的。

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.