Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of death worldwide. Due to latent liver disease, late diagnosis, and nonresponse to systemic treatments, surgical resection and/or biopsy specimens are still generally considered as the gold standard by clinicians for clinical decision-making until now. Since the conventional tissue biopsy is invasive and contains small tissue samples, it is unable to represent tumor heterogeneity or monitor dynamic tumor progression. Therefore, it is imperative to find a new less invasive or noninvasive diagnostic strategy to detect HCC at an early stage and to monitor HCC recurrence. Over the past years, a new diagnostic concept known as “liquid biopsy” has emerged with substantial attention. Liquid biopsy is noninvasive and allows repeated analyses to monitor tumor recurrence, metastasis or treatment responses in real time. With the advanced development of new molecular techniques, HCC circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) detection have achieved interesting and encouraging results. In this review, we focus on the clinical applications of CTCs and ctDNA as key components of liquid biopsy in HCC patients.

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.

Metallothionein 1 (MT1s) is a family of cysteine-rich proteins with diverse functions such as metal homeostasis, oxidative stress, and carcinogenesis. However, its involvement in hepatocellular carcinoma (HCC) remains not fully understood. We aimed to explore the contribution of the individual member of MT1s to HCC. Its member mRNA levels were determined in cohort 1 of normal (n = 30), cirrhotic (n = 30), peritumoral (n = 135), and HCC (n = 135). In cohort 1, seven of eight members were down-regulated during the transition from normal liver to HCC, and only MT1G and MT1X were correlated with tumor features and outcomes. The MT1X was selected to be further stained in cohort 2 consisting of a series of liver nodules (15 normal livers, 33 cirrhotic livers, 12 dysplastic nodules, 31 HCC, and 9 HCC metastasis), and in cohort 3 (HCC, n = 85). In cohort 2, MT1X immunoreactivity was reduced in HCC and lost in metastatic HCC and showed good diagnostic performance for HCC (AUC = 0.754, 95%IC = 0.659-0.849). In cohort 3, MT1X expression in peritumoral tissues was independent predictor for HCC (recurrence free survival: HR = 0.34, 95%CI = 0.17-0.66; overall survival: HR = 0.32, 95%CI = 0.16-0.60). Moreover, we found that ectopic overexpression of MT1X delayed G1/S progression of cell cycle and promoted apoptosis in HCC cells in vitro, and suppressed tumor growth and lung metastasis in nude mice in vivo. We further demonstrated that MT1X induces cell cycle arrest and apoptosis by inactivating NF-κB signaling in HCC. In conclusion, MT1X may serve as a candidate of prognostic indicator and inhibits the progression and metastasis of HCC.

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