Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of l-type amino acid transporter 1

Abstract

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSION This study provides new perspectives on LAT1-dependent cellular processes and a rationale for therapeutics targeting reprogrammed cancer metabolism.