Amino Acid Transporters ASCT2 and LAT1 in Human Hepatocellular Cancer Growth

描述（由申请人提供）：该项目旨在检查靶向氨基酸转运蛋白ASCT 2和LAT 1在人肝细胞癌（HCC）中的疗效，HCC是一种预后不良且治疗选择有限的恶性肿瘤。ASCT 2和LAT 1在包括HCC在内的广泛的原发性人类癌症中协同上调，但目前尚不清楚是什么使这两种转运蛋白（几种转运蛋白中的一种）如此受恶性肿瘤的青睐。这两种转运蛋白在正常肝细胞（肝细胞）中均不以可检测的水平表达，使其成为有吸引力的治疗靶点。此外，ASCT 2和LAT 1已被证明在人类癌细胞的质膜中物理关联，这表明它们合作以帮助推动癌细胞的生长。我们实验室以前的工作表明，ASCT 2的靶向沉默会导致人HCC细胞的程序性细胞死亡（凋亡）;在这里，我们将LAT 1纳入转运蛋白靶向治疗的全部药物中。目前提出的项目的主要目的是测试靶向转运蛋白治疗在HCC中的广泛适用性，并评估ASCT 2和LAT 1在HCC生长和生存信号中的作用。这一目标将通过使用10种人肝癌细胞系来实现，这些细胞系代表了不同HCC表型的广泛临床相关谱。将在10个肝细胞瘤细胞系的每一个中建立靶向ASCT 2或LAT 1的短发夹RNA（shRNA）的稳定维持的诱导型表达系统，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。将通过蛋白质印迹分析评估转运蛋白沉默对生长和存活信号传导（分别为mTOR和Akt途径）的影响。该项目的第二个目的是确定ASCT 2和LAT 1是否在HCC细胞的质膜中物理关联，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涉及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）诱导转运蛋白复合物转运到质膜，在那里它们发挥作用。在转运蛋白沉默不诱导细胞凋亡的情况下，将通过球体形成（HCC细胞系的三维聚集和生长）评估其在初始无血管生长（新生肿瘤形成）中的作用。总的来说，这些目标将深入了解开发转运蛋白靶向治疗的潜力，以及每个转运蛋白在人类HCC生物学中的作用。公共卫生相关性：该项目旨在研究靶向癌症ASCT 2和LAT 1中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展和生长中的作用。在美国，HCC的发病率正在上升，主要是由于慢性丙型肝炎感染的患病率不断增加，目前这种顽固的恶性肿瘤的治疗选择有限。因此，在这种癌症中找到一个适合治疗的“致命弱点”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.