ObjectiveWe aimed to elucidate the mutual regulation mechanism of ubiquitin-specific protease 22 (USP22) and hypoxia inducible factor-1α (HIF1α), and the mechanism they promote the stemness of hepatocellular carcinoma (HCC) cells under hypoxic conditions.DesignCell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were established to detect the stemness of HCC cells. Immunoprecipitation, ubiquitination assay and chromatin immunoprecipitation assay were used to elucidate the mutual regulation mechanism of USP22 and HIF1α. HCC patient samples and The Cancer Genome Atlas data were used to demonstrate the clinical significance. In vivo USP22-targeting experiment was performed in mice bearing HCC.ResultsUSP22 promotes hypoxia-induced HCC stemness and glycolysis by deubiquitinating and stabilising HIF1α. As direct target genes of HIF1α, USP22 and TP53 can be transcriptionally upregulated by HIF1α under hypoxic conditions. In TP53 wild-type HCC cells, HIF1α induced TP53-mediated inhibition of HIF1α-induced USP22 upregulation. In TP53-mutant HCC cells, USP22 and HIF1α formed a positive feedback loop and promote the stemness of HCC. HCC patients with a loss-of-function mutation at TP53 and high USP22 and/or HIF1α expression tend to have a worse prognosis. The USP22-targeting lipopolyplexes caused high tumour inhibition and high sorafenib sensitivity in mice bearing HCC.ConclusionUSP22 promotes hypoxia-induced HCC stemness by a HIF1α/USP22 positive feedback loop on TP53 inactivation. USP22 is a promising target for the HCC therapy.

Resistance to sorafenib severely hinders its effectiveness against hepatocellular carcinoma (HCC). Cancer stemness is closely connected with resistance to sorafenib. Methods for reversing the cancer stemness remains one of the largest concerns in research and the lack of such methods obstructs current HCC therapeutics. Ubiquitin‐specific protease 22 (USP22) is reported to play a pivotal role in HCC stemness and multidrug resistance (MDR). Herein, a galactose‐decorated lipopolyplex (Gal‐SLP) is developed as an HCC‐targeting self‐activated cascade‐responsive nanoplatform to co‐delivery sorafenib and USP22 shRNA (shUSP22) for synergetic HCC therapy. Sorafenib, entrapped in the Gal‐SLPs, induced a reactive oxygen species (ROS) cascade and triggered rapid shUSP22 release. Thus, Gal‐SLPs dramatically suppressed the expression of USP22. The downregulation of USP22 suppresses multidrug resistance‐associated protein 1 (MRP1) to induce intracellular sorafenib accumulation and hampers glycolysis of HCC cells. As a result, Gal‐SLPs efficiently inhibit the viability, proliferation, and colony formation of HCC cells. A sorafenib‐insensitive patient‐derived xenograft (PDX) model is established and adopted to evaluate in vivo antitumor effect of Gal‐SLPs. Gal‐SLPs exhibit potent antitumor efficiency and biosafety. Therefore, Gal‐SLPs are expected to have great potential in the clinical treatment of HCC.

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Background: The administration of calcineurin inhibitors (CNIs) posttransplant has been implicated as an independent risk factor for the recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT). The new immunosuppressive agent sirolimus (SRL) acts as a primary immunosuppressant or antitumor agent. In this study we investigated the effect of sirolimus-based immunosuppression compared to CNIs (non-SRL) on the outcomes of LT candidates with HCC.Methods: We retrospectively analyzed 204 HCC patients who underwent LT in our hospital between January 2, 2014 and December 10, 2017. The median of the follow-up duration of patients was 24.5 months.The patients were divided into a sirolimus (SRL) group (76 patients) and a non-sirolimus (non-SRL) group (128 patients). Patients exceeding the LT criteria were analyzed as subgroups. Disease-free survival (DFS) and overall survival (OS) after tumor recurrence were compared using the Kaplan-Meier method. Univariate and multivariate Cox analyses were used to compare OS between the SRL and non-SRL groups.Results: The SRL group achieved better OS compared to the non-SRL group, while there was no significant difference in DFS. Subgroup (Milan criteria-based or Hangzhou criteria-based) analyses revealed that patients exceeding, rather than meeting, the Milan or Hangzhou criteria benefited from SRL (exceeding the Milan criteria: P=0.002; exceeding the Hangzhou criteria: P<0.001). There was no significant difference in OS between the SRL group and the non-SRL group that met the Milan or Hangzhou criteria.Conclusions: SRL can improve survival outcomes in LT patients with HCC exceeding the Hangzhou criteria.

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The mammalian target of rapamycin (mTOR) pathway is frequently deregulated and has critical roles in cancer progression. mTOR inhibitor has been widely used in several kinds of cancers and is strongly recommended in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT). However, the poor response to mTOR inhibitors due to resistance remains a challenge. Hypoxia‐associated resistance limits the therapeutic efficacy of targeted drugs. The present study established models of HCC clinical samples and cell lines resistance to mTOR inhibitor sirolimus and screened out E2F7 as a candidate gene induced by hypoxia and promoting sirolimus resistance. E2F7 suppressed mTOR complex 1 via directly binding to the promoter of the TSC1 gene and stabilizes hypoxia‐inducible factor‐1α activating its downstream genes, which are responsible for E2F7‐dependent mTOR inhibitor resistance. Clinically, low E2F7 expression could be an effective biomarker for recommending patients with HCC for anti‐mTOR–based therapies after LT. Targeting E2F7 synergistically inhibited HCC growth with sirolimus in vivo. E2F7 is a promising target to reverse mTOR inhibition resistance. Collectively, our study points to a role for E2F7 in promoting mTOR inhibitor resistance in HCC and emphasizes its potential clinical significance in patients with HCC after LT.

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