Endogenous protein S100A14 stabilizes glutaminase to render hepatocellular carcinoma resistant to sorafenib

Many cases of advanced hepatocellular carcinoma (HCC) are resistant to the widely used drug sorafenib, which worsens prognosis. While many studies have explored how acquired resistance emerges during drug exposure, the mechanism underlying primary resistance before treatment still remain elusive. Here, we performed single-cell lineage tracing and RNA sequencing to identify sorafenib-resistant lineages in HCC, and demonstrated that high expression of S100A14 was positively associated with primary sorafenib resistance. Knocking down S100A14 rendered xenograft tumors in mice significantly more sensitive to sorafenib. Mechanistic studies indicated that S100A14 binds to glutaminase and blocks its phosphorylation at residues Y308 and S314, which in turn inhibits its ubiquitination and subsequent degradation. This stabilization of glutaminase reduces oxidative stress in HCC cells and thereby antagonizes the ability of sorafenib to induce apoptosis. Inhibiting glutaminase with telaglenastat (CB-839) significantly improved sorafenib efficacy against xenograft tumors in vivo. These results suggest that S100A14 can contribute to primary sorafenib resistance in HCC by stabilizing glutaminase. Thus, analyzing the expression of S100A14 may be useful for predicting primary sorafenib resistance, and inhibiting S100A14 or glutaminase may be effective for preventing or overcoming such resistance. Overall design: We labeled individual cells in drug-naive hepatocellular carcinoma xenografts on mice with randomly unique 14-base pair (bp) DNA sequences called “barcodes”, then exposed the tumors to sorafenib and compared the characteristics of tumor cells before and after drug exposure. The hepatocellular carcinoma cell line Huh7 was transduced with recombinant lentivirus encoding 14 bp barcodes and the fluorescent reporter CopGFP at a sufficiently low multiplicity of infection (MOI) to ensure that most transductants would receive a single barcode.The above cells were injected subcutaneously into NOD/SCID mice and allowed to grow to a volume of approximately 150 mm3, whereupon they were treated for three weeks with sorafenib, which slowed their growth.Two animals were sacrificed before sorafenib treatment and four animals were sacrificed at the end of treatment, their tumors (drug-naive tumors (DN) and drug-treated tumors (DT), respectively) were excised and dissociated, viable CopGFP-positive tumor cells were selected using a flow cytometric sorter,and the DNA barcodes as well as transcriptome in those cells were sequenced.