Molecular mechanisms of therapy resistance in HCC: USP29-mediated HIF1a stabilization promotes Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis and YAP/TAZ and ATF4 collaboratively drive resistance to Sorafenib therapy in hepatocellular carcinoma by preventing ferroptosis

Primary liver cancer is the 6th most common cancer and 4th leading cause of cancer-related death with over 780,000 new cases annually worldwide. Hepatocellular carcinoma (HCC) represents the most common type of primary malignant liver tumor and accounts for 90% of all liver cancers. But only 30% of all HCC patients are diagnosed at the early stage, most of the HCC patients are diagnosed at very advanced stage, when surgical resection, liver transplantation and percutaneous tumor ablation are not applicable. In 2007, Sorafenib was approved by the FDA as the first-line systemic treatment for advanced HCC patients, however, the prolonged median overall survival is only around 3 months, and resistance to Sorafenib often develops very fast in HCC patients. Therefore, the delineation of the detailed mechanisms of how HCC cells respond to Sorafenib will not only help us to improve the efficacy of Sorafenib therapy in HCC patients, but will also be critical to overcome the development of therapy resistance.
To uncover the molecular mechanisms driving the response and resistance to Sorafenib in HCC cells, we first established Sorafenib-resistant HCC cell lines with treatment of either increasing concentration or constant high concentration of Sorafenib on two Sorafenib-susceptible HCC cell lines Huh7 and Hep3B in vitro. Transcriptomic analysis of the established Sorafenib-resistant cells in comparison with their Sorafenib-responsive counterparts were conducted to identify the genes and pathways underlying the development of Sorafenib-resistance in HCC cells.
In a first project, I employed pathway analysis of established Sorafenib-resistant cells and found that HIF1 signaling was upregulated in Sorafenib-resistant HCC cells. As a well-known oncogene, HIF1 has been proved to upregulate drug resistance in many types of cancers, including Sorafenib resistance in HCC. But how HIF1 is regulated and activated in Sorafenib-resistant HCC remains unclear. To uncover the regulation of HIF1 in Sorafenib-resistant HCC, I performed a small-scale siRNA screen targeting different deubiquitylating enzymes (DUBs) in an intrinsic Sorafenib-resistant HCC cell line HLE which identified the DUB USP29 as the potential upstream regulator of HIF1 protein stability in therapy-resistant HCC. Further studies validated that the regulation of HIF1 by USP29 was through deubiquitylation of HIF1. As a consequence, USP29-stabilized HIF1 promoted high glycolysis levels in Sorafenib-resistant HCC cells. Together, the results indicate that USP29 could be a potential biomarker for the prediction of therapy response in HCC patients and highlight the USP29-HIF1-glycolysis regulatory network as an emerging therapeutic target to overcome therapy resistance in HCC patients.
In a second project, I set out to identify potential novel therapeutic targets in HCC to overcome Sorafenib resistance. To achieve this, I performed a shRNA-based genome-wide synthetic lethality screen on Sorafenib-resistant HCC cells. Among several genes, this screen identified the Hippo signaling transcription factor WWTR1, also known as TAZ, as critical in providing Sorafenib-resistant HCC cells with the ability to overcome this therapy. As a functional homologue of TAZ, YAP was also considered as a synthetic lethal gene. Transcriptomic analysis of YAP/TAZ-deficient HCC cells revealed SLC7A11, the gene encoding a cystine importer required for glutathione synthesis, as a potential downstream target of YAP/TAZ transcriptional activity. Gene set enrichment analysis (GSEA) of the expression of YAP/TAZ-deficient cells and the synthetic lethal screening hits indicated that the YAP/TAZ-SLC7A11 axis was activated in Sorafenib-resistant HCC cells to overcome cell death by ferroptosis. Further studies revealed that the regulation of SLC7A11 expression by YAP/TAZ was further regulated by ATF4-dependent and independent mechanisms. Further experimental evidence indicated that a combination treatment of glutathione synthesis inhibitors and Sorafenib was able to re-install ferroptosis in Sorafenib-resistant HCC cells, thus offering a promising new therapeutic approach to overcome Sorafenib resistance in HCC.
In summary, my PhD work generated novel insights into the molecular mechanisms underlying Sorafenib resistance in HCC. 1. As a positive regulator of HIF1, USP29 promotes its stability and transcriptional activation in HCC, thus conferring high glycolysis levels and resistance to Sorafenib in HCC. 2. YAP/TAZ and ATF4 exert critical roles in the regulation of ferroptosis in Sorafenib-resistant HCC by upregulating SLC7A11 expression which re-installs Sorafenib-induced ferroptosis of HCC cells. The findings thus have direct implications on potential therapeutic approaches of how to overcome the development of resistance to Sorafenib, one of the main therapies of advanced HCC in patients.