Cancer-upregulated l-type amino acid transporter 1 (LAT1; SLC7A5) supplies essential amino acids to cancer cells. LAT1 substrates are not only needed for cancer rapid growth, but involved in cellular signaling. LAT1 has been proposed as a potential target for cancer treatment—its inhibitor, JPH203, is currently in clinical trials and targets biliary tract cancer (BTC). Here, we revealed to what extent LAT1 inhibitor affects intracellular amino acid content and what kind of cellular signals are directly triggered by LAT1 inhibition. Liquid chromatography assay combined with o-phthalaldehyde- and 9-fluorenyl-methylchloroformate-based derivatization revealed changes in intracellular amino acid levels induced by LAT1 inhibition with JPH203 treatment in three BTC cell lines. Tandem mass tag-based quantitative phosphoproteomics characterized the effect of JPH203 treatment on BTC cells, and suggested key regulators in LAT1-inhibited cells. We further studied one of the key regulators, CK2 protein kinase, by using Western blot, enzymatic activity assay, and co-immunoprecipitation. We evaluated anticancer effects of combination of JPH203 with CK2 inhibitor using cell growth and would healing assay. JPH203 treatment decreased intracellular levels of LAT1 substrates including essential amino acids of three BTC cell lines, immediately and drastically. We also found levels of some of these amino acids were partially recovered after longer-time treatment. Therefore, we performed phosphoproteomics with short-time JPH203 treatment prior to the cellular compensatory response, and revealed hundreds of differentially phosphorylated sites. Commonly downregulated phosphorylation sites were found on proteins involved in the cell cycle and RNA splicing. Our phosphoproteomics also suggested key regulators immediately responding to LAT1 inhibition. Focusing on one of these regulators, protein kinase CK2, we revealed LAT1 inhibition decreased phosphorylation of CK2 substrate without changing CK2 enzymatic activity. Furthermore, LAT1 inhibition abolished interaction between CK2 and its regulatory protein NOLC1, which suggests regulatory mechanism of CK2 substrate protein specificity controlled by LAT1 inhibition. Moreover, we revealed that the combination of JPH203 with CK2 inhibitor resulted in the enhanced inhibition of proliferation and migration of BTC cells. This study provides new perspectives on LAT1-dependent cellular processes and a rationale for therapeutics targeting reprogrammed cancer metabolism.

中文翻译：

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磷酸化蛋白质组学显示细胞信号立即响应由抑制 l 型氨基酸转运蛋白 1 诱导的癌症氨基酸稳态破坏

癌症上调的 l 型氨基酸转运蛋白 1（LAT1；SLC7A5）为癌细胞提供必需氨基酸。LAT1 底物不仅是癌症快速生长所必需的，而且还参与细胞信号传导。LAT1 已被提议作为癌症治疗的潜在靶点——其抑制剂 JPH203 目前正在进行临床试验并靶向胆道癌 (BTC)。在这里，我们揭示了 LAT1 抑制剂在多大程度上影响细胞内氨基酸含量以及 LAT1 抑制直接触发了什么样的细胞信号。液相色谱分析结合基于邻苯二甲醛和 9-芴基-甲基氯甲酸酯的衍生化揭示了三种 BTC 细胞系中 LAT1 抑制和 JPH203 处理诱导的细胞内氨基酸水平的变化。基于串联质量标签的定量磷酸化蛋白质组学表征了 JPH203 处理对 BTC 细胞的影响，并提出了 LAT1 抑制细胞中的关键调节因子。我们通过使用蛋白质印迹、酶活性测定和共免疫沉淀进一步研究了关键调节剂之一 CK2 蛋白激酶。我们使用细胞生长和愈合试验评估了 JPH203 与 CK2 抑制剂组合的抗癌作用。JPH203 处理可立即显着降低三种 BTC 细胞系的 LAT1 底物（包括必需氨基酸）的细胞内水平。我们还发现其中一些氨基酸的水平在经过较长时间的治疗后得到了部分恢复。因此，我们在细胞代偿反应之前用短时间 JPH203 处理进行了磷酸化蛋白质组学，并揭示了数百个差异磷酸化位点。在参与细胞周期和 RNA 剪接的蛋白质上发现了通常下调的磷酸化位点。我们的磷酸化蛋白质组学还表明关键监管机构会立即响应 LAT1 抑制。着眼于这些调节剂之一，蛋白激酶 CK2，我们发现 LAT1 抑制降低了 CK2 底物的磷酸化，而不改变 CK2 酶活性。此外，LAT1 抑制消除了 CK2 与其调节蛋白 NOLC1 之间的相互作用，这表明 CK2 底物蛋白特异性的调节机制受 LAT1 抑制控制。此外，我们发现 JPH203 与 CK2 抑制剂的组合导致 BTC 细胞增殖和迁移的增强抑制。