Risk versus Benefit of Tyrosine Kinase Inhibitors for Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

Although the treatment landscape has rapidly evolved over the last years, hepatocellular carcinoma (HCC) is one of the most lethal cancers. With recent advances, both immunotherapy and tyrosine kinase inhibitors (TKIs)-based chemotherapy constitute the standard treatment for advanced HCC. A systematic search of randomized clinical trials employing TKIs was performed in 17 databases, obtaining 25 studies evaluating the prognosis, tumor response, and presence of adverse events (AEs) related to TKIs in HCC. Overall effect sizes were estimated for the hazard ratios (HR) and odds ratios (OR) with 95% confidence interval (CI), either extracted or calculated with the Parmar method, employing STATA 16. Heterogeneity was assessed by Chi-square-based Q-test and inconsistency (I2) statistic; source of heterogeneity by meta-regression and subgroup analysis; and publication bias by funnel plot asymmetry and Egger's test. The research protocol was registered in PROSPERO (CRD42023397263). Meta-analysis revealed a correlation between survival and tumor response parameters and TKI treatment vs. placebo, despite detecting high heterogeneity. Combined TKI treatment showed a significantly better objective response rate (ORR) with no heterogeneity, whereas publication bias was only detected with time to progression (TTP). Few gastrointestinal and neurological disorders were associated with TKI treatment vs. placebo or with combined treatment. However, a higher number of serious AEs were related to TKI treatment vs. sorafenib alone. Results show positive clinical benefits from TKI treatment, supporting the approval and maintenance of TKI-based therapy for advanced HCC, while establishing appropriate strategies to maximize efficacy and minimize toxicity.

Correction: Méndez-Blanco et al. Stabilization of Hypoxia-Inducible Factors and BNIP3 Promoter Methylation Contribute to Acquired Sorafenib Resistance in Human Hepatocarcinoma Cells. Cancers 2019, 11, 1984.

In the original publication [...].

Neuropilins as potential biomarkers in hepatocellular carcinoma: a systematic review of basic and clinical implications.

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers worldwide and is characterized by complex molecular carcinogenesis. Neuropilins (NRPs) NRP1 and NRP2 are the receptors of multiple proteins involved in key signaling pathways associated with tumor progression. We aimed to systematically review all the available findings on their role in HCC. We searched the Scopus, Web of Science (WOS), PubMed, Cochrane and Embase databases for articles evaluating NRPs in preclinical or clinical HCC models. This study was registered in PROSPERO (CRD42022349774) and include 49 studies. Multiple cellular and molecular processes have been associated with one or both NRPs, indicating that they are potential diagnostic and prognostic biomarkers in HCC patients. Mainly NRP1 has been shown to promote tumor cell survival and progression by modulating several signaling pathways. NRPs mainly regulate angiogenesis, invasion and migration and have shown to induce invasion and metastasis. They also regulate the immune response and tumor microenvironment, showing a crucial interplay with the hypoxia response and microRNAs in HCC. Altogether, NRP1 and NRP2 are potential biomarkers and therapeutic targets, providing novel insight into the clinical landscape of HCC patients.

Hepatocellular carcinoma cells loss lenvatinib efficacy in vitro through autophagy and hypoxia response-derived neuropilin-1 degradation.

Despite pharmacological advances such as lenvatinib approval, therapeutic failure of hepatocellular carcinoma (HCC) remains a big challenge due to the complexity of its underlying molecular mechanisms. Neuropilin-1 (NRP1) is a co-receptor involved in several cellular processes associated to chemoresistance development. Since both the double-edged process of autophagy and hypoxia-derived response play crucial roles in the loss of therapeutic effectiveness, herein we investigated the interplay among NRP1, autophagy and hypoxia in development of lenvatinib resistance in HCC cell lines. We first analyzed NRP1 expression levels in human HCC samples from public databases, found significantly increased NRP1 expression in human HCC samples as well as its correlation with advanced tumor and metastasis stages. Among 3 HCC cell lines (HepG2, Huh-7 and Hep3B), Hep3B and Huh-7 cells showed significantly increased NRP1 expression levels and cell migration ability together with higher susceptibility to lenvatinib. We demonstrated that NRP1 gene silencing significantly enhanced the anticancer effects of lenvatinib on Hep3B and Huh-7 cells. Furthermore, lenvatinib suppressed NRP1 expression through promoting autophagy in Hep3B and Huh-7 cells; co-treatment with bafilomycin A1 attenuated the antitumor effects of lenvatinib, and NRP1 silencing prevented this loss of in vitro effectiveness of lenvatinib even in the presence of bafilomycin A1. In addition, exposure to a hypoxic microenvironment significantly decreased NRP1 expression through autophagy in Hep3B and Huh-7 cells. Under hypoxia, HIF-1α directly modulated NRP1 expression; HIF-1α silencing not only enhanced the anticancer effects of combined lenvatinib and hypoxia, but also prevented the loss of effectiveness caused by bafilomycin A1, highlighting the potential role of HIF-1α-derived hypoxia response in the adaptive cellular response to lenvatinib and promoting resistance acquisition by autophagy modulation. Overall, NRP1 may constitute a potential therapeutic target to prevent lenvatinib failure derived from a hypoxia-associated modulation of autophagy in advanced HCC.

Neuropilin-1 as a Potential Biomarker of Prognosis and Invasive-Related Parameters in Liver and Colorectal Cancer: A Systematic Review and Meta-Analysis of Human Studies.

Neuropilin-1 (NRP1) is a transmembrane protein involved in numerous cellular functions which has had increasing interest from cancer researchers. Liver cancer and colorectal cancer (CRC) are two of the most frequent and deadly tumors with a complex pharmacological framework. Here, we assessed the prognostic, diagnostic and clinicopathological value of NRP1 in liver cancer and CRC patients. We searched PubMed, Scopus, Web of Science, Embase and Cochrane Library databases for articles evaluating the NRP1 correlation with survival parameters, tumor development or clinicopathological features. Hazard ratios and odds ratios with 95% confidence intervals were extracted or estimated by Parmar method and pooled to evaluate the overall effect size with STATA 16 software. Heterogeneity was analyzed by chi-square-based Q test and I2 statistic, along with meta-regression and subgroup analysis, and publication bias was assessed by funnel plot asymmetry and Egger's test. The study protocol was registered in PROSPERO (CRD42022307062). NRP1 overexpression was significantly correlated with lower survival in liver cancer patients and with tumor development in hepatocarcinoma patients, and was strongly correlated with an increased risk of vascular invasion in liver cancer and metastasis in CRC and liver tumors. These results support the role of NRP1 as a potential and useful biomarker in both types of cancer.

Association of FOXO3 Expression with Tumor Pathogenesis, Prognosis and Clinicopathological Features in Hepatocellular Carcinoma: A Systematic Review with Meta-Analysis.

Forkhead box O3 (FOXO3), an essential transcription factor related to liver disease, has been linked to cancer progression. The most frequent primary liver tumor, hepatocellular carcinoma (HCC), has an elevated mortality rate and patient outcomes remain very poor. Here, we examined the diagnostic, prognostic and clinicopathological significance of FOXO3 expression in HCC. We systematically searched Cochrane, Embase, PubMed, Scopus and Web of Science. Articles analyzing FOXO3 levels in HCC patient samples and its relationship with tumor development, survival or clinicopathological factors were selected. Hazard ratios, odds ratios and 95% confidence intervals were extracted, estimated by Parmar method or calculated and pooled across studies. Heterogeneity was evaluated by chi-square-based Q and I2 tests, while publication bias by funnel plots and Egger's test. Subgroup analysis was performed when heterogeneity was evident. The study protocol was registered in PROSPERO (CRD42021237321), and data were meta-analyzed employing STATA 16. Five studies involving 1059 HCC cases were finally included in this meta-analysis, finding that high FOXO3 levels significantly correlate with HCC development and shorter overall survival. Moreover, subgroup analysis revealed a significant association between positive FOXO3 expression and the risk of invasion. Thus, FOXO3 could function as a novel biomarker with diagnostic and prognostic value in HCC.

Autophagy-Related Chemoprotection against Sorafenib in Human Hepatocarcinoma: Role of FOXO3 Upregulation and Modulation by Regorafenib.

Early acquisition of sorafenib resistance is responsible for the dismal prognosis of advanced hepatocarcinoma (HCC). Autophagy, a catabolic process involved in liver homeostasis, has been associated with chemosensitivity modulation. Forkhead box O3 (FOXO3) is a transcription factor linked to HCC pathogenesis whose role on autophagy-related sorafenib resistance remains controversial. Here, we unraveled the linkage between autophagy and sorafenib resistance in HCC, focusing on the implication of FOXO3 and its potential modulation by regorafenib. We worked with two HepG2-derived sorafenib-resistant HCC in vitro models (HepG2S1 and HepG2S3) and checked HCC patient data from the UALCAN database. Resistant cells displayed an enhanced basal autophagic flux compared to HepG2, showing higher autophagolysosome content and autophagy markers levels. Pharmacological inhibition of autophagy boosted HepG2S1 and HepG2S3 apoptosis and subG1 cells, but reduced viability, indicating the cytoprotective role of autophagy. HCC samples displayed higher FOXO3 levels, being associated with shorter survival and autophagic genes expression. Consistently, chemoresistant in vitro models showed significant FOXO3 upregulation. FOXO3 knockdown suppressed autophagy and caused resistant cell death, demonstrating that overactivation of such pro-survival autophagy during sorafenib resistance is FOXO3-dependent; a cytoprotective mechanism that the second-line drug regorafenib successfully abolished. Therefore, targeting FOXO3-mediated autophagy could significantly improve the clinical efficacy of sorafenib.

Prognostic and clinicopathological significance of hypoxia-inducible factors 1α and 2α in hepatocellular carcinoma: a systematic review with meta-analysis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly recurrent tumor after resection and has been closely related to hypoxia. Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) have been shown to contribute to tumor progression and therapy resistance in HCC. We evaluated the prognostic and clinicopathological significance of HIF-1α and HIF-2α in HCC patients. METHODS: We systematically searched Embase, Cochrane, PubMed, Scopus and Web of Science (WOS) from inception to 1 June 2020 for studies evaluating HIF-1α and/or HIF-2α expression in HCC. Selected articles evaluate at least one factor by immunohistochemistry (IHC) in HCC patients who underwent surgical resection, and its relationship with prognosis and/or clinicopathological features. Study protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO; CDR42020191977). We meta-analyzed the data extracted or estimated according to the Parmar method employing STATA software. We evaluated the overall effect size for the hazard ratio (HR) and odds ratio (OR) with 95% confidence interval (CI), as well as heterogeneity across studies with the I 2 statistic and chi-square-based Q test. Moreover, we conducted subgroup analysis when heterogeneity was substantial. Publication bias was assessed by funnel plot asymmetry and Egger's test. RESULTS: HIF-1α overexpression was correlated with overall survival (OS), disease-free survival (DFS)/recurrence-free survival (RFS) and clinicopathological features including Barcelona Clinic Liver Cancer (BCLC), capsule infiltration, intrahepatic metastasis, lymph node metastasis, tumor-node-metastasis (TNM), tumor differentiation, tumor number, tumor size (3 cm), vascular invasion and vasculogenic mimicry. We also detected a possible correlation of HIF-1α with alpha-fetoprotein (AFP), cirrhosis, histological grade, tumor size (5 cm) and albumin after subgroup analysis. Initially, only DFS/RFS appeared to be associated with HIF-2α overexpression. Subgroup analysis denoted that HIF-2α overexpression was related to OS and capsule infiltration. CONCLUSIONS: HIF-1α and HIF-2α overexpression is related to poor OS, DFS/RFS and some clinicopathological features of HCC patients, suggesting that both factors could be useful HCC biomarkers.

Melatonin as an Antitumor Agent against Liver Cancer: An Updated Systematic Review.

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine with antioxidant, chronobiotic and anti-inflammatory properties; reduced levels of this hormone are associated with higher risk of cancer. Several beneficial effects of melatonin have been described in a broad number of tumors, including liver cancers. In this work we systematically reviewed the publications of the last 15 years that assessed the underlying mechanisms of melatonin activities against liver cancers, and its role as coadjuvant in the treatment of these tumors. Literature research was performed employing PubMed, Scopus and Web of Science (WOS) databases and, after screening, 51 articles were included. Results from the selected studies denoted the useful actions of melatonin in preventing carcinogenesis and as a promising treatment option for the primary liver tumors hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), either alone or in combination with other compounds. Different processes were modulated by the indole, such as inhibition of oxidative stress, proliferation, angiogenesis and invasion, promotion of immune system response, cell cycle arrest and apoptosis, as well as recovery of circadian rhythms and autophagy modulation. Taken together, the present systematic review highlights the evidence that document the potential role of melatonin in improving the landscape of liver tumor treatment.

Stabilization of Hypoxia-Inducible Factors and BNIP3 Promoter Methylation Contribute to Acquired Sorafenib Resistance in Human Hepatocarcinoma Cells.

Despite sorafenib effectiveness against advanced hepatocarcinoma (HCC), long-term exposure to antiangiogenic drugs leads to hypoxic microenvironment, a key contributor to chemoresistance acquisition. We aimed to study the role of hypoxia in the development of sorafenib resistance in a human HCC in vitro model employing the HCC line HepG2 and two variants with acquired sorafenib resistance, HepG2S1 and HepG2S3, and CoCl2 as hypoximimetic. Resistant cells exhibited a faster proliferative rate and hypoxia adaptive mechanisms, linked to the increased protein levels and nuclear translocation of hypoxia-inducible factors (HIFs). HIF-1α and HIF-2α overexpression was detected even under normoxia through a deregulation of its degradation mechanisms. Proapoptotic markers expression and subG1 population decreased significantly in HepG2S1 and HepG2S3, suggesting evasion of sorafenib-mediated cell death. HIF-1α and HIF-2α knockdown diminished resistant cells viability, relating HIFs overexpression with its prosurvival ability. Additionally, epigenetic silencing of Bcl-2 interacting protein 3 (BNIP3) was observed in sorafenib resistant cells under hypoxia. Demethylation of BNIP3 promoter, but not histone acetylation, restored BNIP3 expression, driving resistant cells' death. Altogether, our results highlight the involvement of HIFs overexpression and BNIP3 methylation-dependent knockdown in the development of sorafenib resistance in HCC. Targeting both prosurvival mechanisms could overcome chemoresistance and improve future therapeutic approaches.

Antitumor Effects of Quercetin in Hepatocarcinoma In Vitro and In Vivo Models: A Systematic Review.

Quercetin is a flavonoid present in fruits, vegetables and plants with antioxidant, anti-inflammatory and anticancer properties. Its beneficial activities have been demonstrated in different human pathologies, including hepatoprotective effects against liver disorders. High mortality and late diagnosis of the primary liver tumor hepatocarcinoma (HCC) makes this cancer an interesting target for the study of quercetin effects. Our aim was to systematically review antitumor activities of quercetin in HCC preclinical studies employing single, encapsulated, combined or derived quercetin forms. Literature search was conducted in PubMed, Scopus and Web of Science (WOS), and 39 studies were finally included. We found that 17 articles evaluated quercetin effects alone, six used encapsulated strategy, 10 combined this flavonoid, two decided to co-encapsulate it and only four studied effects of quercetin derivatives, highlighting that only nine included in vivo models. Results evidence the quercetin antiproliferative and proapoptotic properties against HCC either alone and with the mentioned strategies; nevertheless, few investigations assessed specific activities on different processes related with cancer progression. Overall, further studies including animal models are needed to deeper investigate the precise mechanisms of action of quercetin as antitumor agent, as well as the potential of novel strategies aimed to improve quercetin effects in HCC.

Anti-tumoral activity of single and combined regorafenib treatments in preclinical models of liver and gastrointestinal cancers.

Regorafenib is a sorafenib-derived chemotherapy drug belonging to the multikinase inhibitor family. This agent effectively targets a wide range of tyrosine kinases involved in cancer biology, such as those implicated in oncogenesis, angiogenesis, and tumor microenvironment control. The beneficial effects of regorafenib in clinical trials of patients who suffer from advanced hepatocellular carcinoma (HCC), colorectal cancer (CRC) or gastrointestinal stromal tumors (GISTs) refractory to standard treatments led to regorafenib monotherapy approval as a second-line treatment for advanced HCC and as a third-line treatment for advanced CRC and GISTs. Multiple in vitro and in vivo studies have been performed over the last decade to reveal the molecular mechanisms of the favorable actions exerted by regorafenib in patients. Given the hypothetical loss of sensitivity to regorafenib in tumor cells, preclinical research is also searching for novel therapeutic approaches consisting of co-administration of this drug plus other agents as a strategy to improve regorafenib effectiveness. This review summarizes the anti-tumor effects of regorafenib in single or combined treatment in preclinical models of HCC, CRC and GISTs and discusses both the global and molecular effects that account for its anti-cancer properties in the clinical setting.

Sorafenib resistance in hepatocarcinoma: role of hypoxia-inducible factors.

Sorafenib, a multikinase inhibitor with antiproliferative, antiangiogenic, and proapoptotic properties, constitutes the only effective first-line drug approved for the treatment of advanced hepatocellular carcinoma (HCC). Despite its capacity to increase survival in HCC patients, its success is quite low in the long term owing to the development of resistant cells through several mechanisms. Among these mechanisms, the antiangiogenic effects of sustained sorafenib treatment induce a reduction of microvessel density, promoting intratumoral hypoxia and hypoxia-inducible factors (HIFs)-mediated cellular responses that favor the selection of resistant cells adapted to the hypoxic microenvironment. Clinical data have demonstrated that overexpressed HIF-1α and HIF-2α in HCC patients are reliable markers of a poor prognosis. Thus, the combination of current sorafenib treatment with gene therapy or inhibitors against HIFs have been documented as promising approaches to overcome sorafenib resistance both in vitro and in vivo. Because the depletion of one HIF-α subunit elevates the expression of the other HIF-α isoform through a compensatory loop, targeting both HIF-1α and HIF-2α would be a more interesting strategy than therapies that discriminate among HIF-α isoforms. In conclusion, there is a marked correlation between the hypoxic microenvironment and sorafenib resistance, suggesting that targeting HIFs is a promising way to increase the efficiency of treatment.

Melatonin enhances sorafenib actions in human hepatocarcinoma cells by inhibiting mTORC1/p70S6K/HIF-1α and hypoxia-mediated mitophagy.

The antiangiogenic effects of sustained sorafenib treatment in hepatocellular carcinoma (HCC) lead to the occurrence of hypoxia-mediated drug resistance resulting in low therapy efficiency and negative outcomes. Combined treatments with coadjuvant compounds to target the hypoxia-inducible factor-1α (HIF-1α) represent a promising therapeutic approach through which sorafenib efficiency may be improved. Melatonin is a well-documented oncostatic agent against different cancer types. Here, we evaluated whether melatonin could enhance sorafenib cytotoxicity and overcome the hypoxia-mediated resistance mechanisms in HCC. The pharmacological melatonin concentration (2 mM) potentiated the oncostatic effects of sorafenib (5 µM) on Hep3B cells even under hypoxia. Melatonin downregulated the HIF-1α protein synthesis through the inhibition of the mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase beta-1 (p70S6K)/ribosomal protein S6 (RP-S6) pathway, although the indole enhanced Akt phosphorylation by the mTORC1/C2 negative feedback. Furthermore, melatonin and sorafenib coadministration reduced the HIF-1α-mitophagy targets expression, impaired autophagosome formation and subsequent mitochondria and lysosomes colocalization. Together, our results indicate that melatonin improves the Hep3B sensitivity to sorafenib, preventing HIF-1α synthesis to block the cytoprotective mitophagy induced by the hypoxic microenvironment, an important element of the multifactorial mechanisms responsible for the chemotherapy failure.

Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy.

Effects of sorafenib in hepatocellular carcinoma (HCC) are frequently transient due to tumor-acquired resistance, a phenotype that could be targeted by other molecules to reduce this adaptive response. Because melatonin is known to exert antitumor effects in HCC cells, this study investigated whether and how melatonin reduces resistance to sorafenib. Susceptibility to sorafenib (10 nmol/L to 50 µmol/L) in the presence of melatonin (1 and 2 mmol/L) was assessed in HCC cell lines HepG2, HuH7, and Hep3B. Cell viability was reduced by sorafenib from 1 µmol/L in HepG2 or HuH7 cells, and 2.5 µmol/L in Hep3B cells. Co-administration of melatonin and sorafenib exhibited a synergistic cytotoxic effect on HepG2 and HuH7 cells, while Hep3B cells displayed susceptibility to doses of sorafenib that had no effect when administrated alone. Co-administration of 2.5 µmol/L sorafenib and 1 mmol/L melatonin induced apoptosis in Hep3B cells, increasing PARP hydrolysis and BAX expression. We also observed an early colocalization of mitochondria with lysosomes, correlating with the expression of mitophagy markers PINK1 and Parkin and a reduction of mitofusin-2 and mtDNA compared with sorafenib administration alone. Moreover, increased reactive oxygen species production and mitochondrial membrane depolarization were elicited by drug combination, suggesting their contribution to mitophagy induction. Interestingly, Parkin silencing by siRNA to impair mitophagy significantly reduced cell killing, PARP cleavage, and BAX expression. These results demonstrate that the pro-oxidant capacity of melatonin and its impact on mitochondria stability and turnover via mitophagy increase sensitivity to the cytotoxic effect of sorafenib.