The only first-line treatment approved for advanced hepatocellular carcinoma (HCC) is sorafenib. Since many patients experience drug resistance, the discovery of more effective therapeutic strategies represents an unmet clinical need. MicroRNA (MiR)-122 is downregulated in most HCCs, while oncogenic SerpinB3 is upregulated. Here, we assessed the relationship between miR-122 and SerpinB3 and their influence on cell phenotype and sorafenib resistance in HCC. A bioinformatics analysis identified SerpinB3 among hypothetical miR-122 targets. In SerpinB3-overexpressing HepG2 cells, miR-122 transfection decreased SerpinB3 mRNA and protein levels, whereas miR-122 inhibition increased SerpinB3 expression. Luciferase assay demonstrated the interaction between miR-122 and SerpinB3 mRNA. In an HCC rat model, high miR-122 levels were associated with negative SerpinB3 expression, while low miR-122 levels correlated with SerpinB3 positivity. A negative correlation between miR-122 and SerpinB3 or stem cell markers was found in HCC patients. Anti-miR-122 transfection increased cell viability in sorafenib-treated Huh-7 cells, while miR-122 overexpression increased sorafenib sensitivity in treated cells, but not in those overexpressing SerpinB3. In conclusion, we demonstrated that miR-122 targets SerpinB3, and its low levels are associated with SerpinB3 positivity and a stem-like phenotype in HCC. MiR-122 replacement therapy in combination with sorafenib deserves attention as a possible therapeutic strategy in SerpinB3-negative HCCs.

Background: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2α-dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1α and HIF-2α. SB3 acts by (i) up-regulating HIF-1α transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2α stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2α transcripts; (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2α transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2α transcripts. Conclusions: These data outline either HIF-2α and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.

Hypoxia and hypoxia-inducible factors (HIFs) are involved in chronic liver disease progression. We previously showed that hepatocyte HIF-2α activation contributed significantly to nonalcoholic fatty liver disease progression in experimental animals and human patients. In this study, using an appropriate genetic murine model, we mechanistically investigated the involvement of hepatocyte HIF-2α in experimental nonalcoholic steatohepatitis (NASH)-related carcinogenesis. The role of HIF-2α was investigated by morphologic, cellular, and molecular biology approaches in the following: (1) mice carrying hepatocyte-specific deletion of HIF-2α (HIF-2α^-/- mice) undergoing a NASH-related protocol of hepatocarcinogenesis; (2) HepG2 cells stably transfected to overexpress HIF-2α; and (3) liver specimens from NASH patients with hepatocellular carcinoma. Mice carrying hepatocyte-specific deletion of HIF-2α (hHIF-2α^-/-) showed a significant decrease in the volume and number of liver tumors compared with wild-type littermates. These effects did not involve HIF-1α changes and were associated with a decrease of cell proliferation markers proliferating cell nuclear antigen and Ki67. In both human and rodent nonalcoholic fatty liver disease–related tumors, HIF-2α levels were strictly associated with hepatocyte production of SerpinB3, a mediator previously shown to stimulate liver cancer cell proliferation through the Hippo/Yes-associated protein (YAP)/c-Myc pathway. Consistently, we observed positive correlations between the transcripts of HIF-2α, YAP, and c-Myc in individual hepatocellular carcinoma tumor masses, while HIF-2α deletion down-modulated c-Myc and YAP expression without affecting extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and AKT-dependent signaling. In vitro data confirmed that HIF-2α overexpression induced HepG2 cell proliferation through YAP-mediated mechanisms. These results indicate that the activation of HIF-2α in hepatocytes has a critical role in liver carcinogenesis during NASH progression, suggesting that HIF-2α–blocking agents may serve as novel putative therapeutic tools.

Background: In the setting of liver injury hepatic progenitor cells are activated, counterbalancing the inhibited regenerative capacity of mature hepatocytes. Chronic activation of this compartment may give rise to a subset of liver tumours with poor prognosis. SerpinB3, a serpin over-expressed in injured liver and in primary liver cancer, has been shown to induce apoptosis resistance, epithelial to mesenchymal transition and to increase TGF-beta and Myc expression. Aim of the present study was to explore the presence of SerpinB3 in hepatic progenitor cells in human livers and in a mouse model of liver stem/progenitor cell activation. Hepatic progenitor cells were analysed in foetal and adult livers at protein and transcriptional levels. To induce experimental activation of the liver stem/progenitor compartment, C57BL/6J mice were injected with lipopolysaccharide plus D-galactosamine and were sacrificed at different time points. Liver cDNA was amplified using specific primers for mouse-homologous SerpinB3 isoforms and automatically sequenced. Results: The presence of SerpinB3 in the progenitor cell compartment was detected in sorted human foetal and adult epithelial cell adhesion molecule (EpCAM) positive liver cells. By immunohistochemistry SerpinB3 was found in human cirrhotic livers in portal areas with progenitor cell activation showing ductular proliferation. CK-7, CK-19, EpCAM and CD-90 positive cell were also positive for SerpinB3. In the animal model, time course analysis in liver specimens revealed a progressive increase of SerpinB3 and a parallel decrease of activated caspase 3, which was barely detectable at 20 hours. Transcription analysis confirmed the presence of SerpinB3-homologous only in the liver of injured mice and sequence analysis proved its belonging to mouse Serpinb3b. Conclusion: SerpinB3 is highly expressed in hepatic stem/progenitor cell compartment of both foetal and adult livers.

Foot ulcerations are a disabling complication of diabetes and no treatment is currently available based on disease mechanisms. The protein serpin B3 (SB3) was identified as a positive biomarker of successful diabetic wound healing; therefore, its exogenous administration may promote healing. The topical administration of SB3 is challenging due to its protein nature. Physical entrapment in wet sol–gel silica can stabilize the protein’s conformation and permit its sustained delivery. However, irreversible syneresis and poor viscoelastic properties hamper wet sol–gel silica application as a semisolid vehicle. To overcome these limits, a sol–gel silica/hydroxypropylmethylcellulose (HPMC) hydrogel blend was developed. SB3 entrapped in 8% SiO₂ wet sol–gel silica preserved its structure, was stabilized against denaturation, and was slowly released for at least three days. Blending a silica gel with an HPMC–glycerol (metolose-G) hydrogel permitted spreadability without affecting the protein’s release kinetics. When administered in vivo, SB3 in silica/metolose-G—but not in solution or in metolose-G alone—accelerated wound healing in SB3 knockout and diabetic mouse models. The results confirmed that SB3 is a new pharmacological option for the treatment of chronic ulcers, especially when formulated in a slow-releasing vehicle. Silica–metolose-G represents a novel type of semisolid dosage form which could also be applied for the formulation of other bioactive proteins.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. NAFLD can evolve from simple fatty liver to non-alcoholic steatohepatitis (NASH), and ultimately, to cirrhosis. Inflammation and oxidative stress, promoted by mitochondrial dysfunction, play a crucial role in the onset and development of NASH. To date, no therapy has been approved for NAFLD and NASH. The aim of this study is to evaluate if the anti-inflammatory activity of acetylsalicylic acid (ASA) and the mitochondria-targeted antioxidant effect of mitoquinone could hinder the progression of non-alcoholic steatohepatitis. In mice, fatty liver was induced through the administration of a deficient in methionine and choline and rich in fat diet. Two experimental groups were treated orally with ASA or mitoquinone. Histopathologic evaluation of steatosis and inflammation was performed; the hepatic expression of genes associated with inflammation, oxidative stress, and fibrosis was evaluated; the protein expression of IL-10, cyclooxygenase 2, superoxide dismutase 1, and glutathione peroxidase 1 in the liver was analyzed; a quantitative analysis of 15-epi-lipoxin A4 in liver homogenates was performed. Mitoquinone and ASA significantly reduced liver steatosis and inflammation by decreasing the expression of TNFα, IL-6, Serpinb3, and cyclooxygenase 1 and 2 and restoring the anti-inflammatory IL-10. Treatment with mitoquinone and ASA increased the gene and protein expression of antioxidants, i.e., catalase, superoxide dismutase 1, and glutathione peroxidase 1, and decreased the expression of profibrogenic genes. ASA normalized the levels of 15-epi-Lipoxin A4. In mice fed with a deficient in methionine and choline and rich in fat diet, mitoquinone and ASA reduce steatosis and necroinflammation and may represent two effective novel strategies for the treatment of non-alcoholic steatohepatitis.