Liver cancer is a global issue, being the most common cancer and the leading cause of cancer death in transition countries. In 2020, almost 9,06,000 patients were diagnosed with liver cancer and over 8,30,000 deaths were documented worldwide. Hepatocellular carcinoma (HCC) has the highest prevalence among the different subtypes of liver cancer (Sung et al., 2021). Viral infections, more specifically hepatitis B and C virus (Datfar et al., 2021), lifestyle factors such as alcohol intake (Matsushita and Takaki, 2019), as well as type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) (Estes et al., 2018) remain the leading risk factors, depending on the region considered. Hepatocellular carcinoma remains one of the most common causes of cancer death, especially in men, and has one of the lowest 5-year survival rates of all different cancer types (Siegel et al., 2022).

In addition to chronic inflammation, tissue remodeling and changes in cellular signaling are pathogenetic factors of carcinogenesis (Refolo et al., 2020). Interestingly, patients with a non-cirrhotic HCC, mostly caused by NAFLD, seem to show a more severe HCC histopathology on one hand but better overall survival on the other hand (Gawrieh et al., 2019).

Limited stages of HCC can be treated with locoregional procedures such as surgical (e.g., resection, transplantation) or radiological (e.g., transarterial chemoembolization, selective internal radiotherapy) intervention, prolonging survival for more than 5 years, depending on the underlying liver disease. Once the disease progresses, survival is compromised to approximately 1 year even with systemic therapy (Forner et al., 2018; Vogel et al., 2022). Systemic therapy options were shown to increase overall survival (OS) and progression-free survival (PFS). However, either applying combination therapies including immune checkpoint inhibitors such as atezolizumab plus bevacizumab (OS 13,4-19,2 months; PFS 6,9 months) (Finn et al., 2020) or tyrosine kinase inhibitors lenvatinib (OS 13,6 months; PFS 7,3 months) (Kudo et al., 2018) or sorafenib (OS 10,7-15,5 months; PFS 3,6-5,5) (Kelley et al., 2022) has not altered the severity or mortality of the disease so far (Vogel et al., 2022).

The first targeted and currently a widely used systemic therapy for HCC is the oral multikinase inhibitor sorafenib binding in an ATP-binding pocket to inhibit kinase function (Hwang et al., 2013), which predominantly inhibits angiogenesis via binding the vascular endothelial growth receptor (VEGFR). Furthermore, it targets the cell proliferation and differentiation via the rapidly accelerated fibrosarcoma (RAF) signaling pathway (Wilhelm et al., 2004) and the platelet-derived growth factor receptor-β (PDGFR-β) (Mody and Abou-Alfa, 2019) as well as the beneficial effects, particularly in cancer and its complications, which is likely due to the inhibition of nuclear factor kappa B (NF-κB) and production of the pro-resolution mediators at the molecular level (Freitas and Campos, 2019).

In addition to the known anti-angiogenic and anti-proliferative effects of sorafenib it has also been described to have effects on the soluble epoxide hydrolase (sEH) which showed similar anti-inflammatory effects as conventional sEH inhibitors in lipopolysaccharide-induced inflammation models in mice (Liu et al., 2009). Sorafenib is a potent inhibitor of sEH compared with conventional urea-based sEH inhibitors (Hwang et al., 2013). The sEH is expressed in numerous human tissues with the main distinction between microsomal epoxide hydrolase (mEH) and sEH (Morisseau and Hammock, 2013). The sEH metabolization is the dominant pathway in humans, so that it can be assumed that sEH inhibition has a stabilizing effect on endogenous epoxy metabolites in tissues (Spector and Kim, 2015).

The epoxidation of long-chain polyunsaturated fatty acids (LC-PUFAs), such as the omega-6 (n-6) PUFA arachidonic acid (C20:4n6, AA), as well as the omega-3 (n-3) PUFAs eicosapentaenoic acid (C20:5n3, EPA) and docosahexaenoic acid (C22:6n3, DHA) to epoxyeicosatrienoic acids (EETs), epoxyeicosatetraenoic acids (EEQs) and epoxydocosapentaenoic acids (EDPs), respectively, are catalyzed by the cytochrome P450 (CYP) epoxygenases (Figure 1). These epoxymetabolites are then further metabolized via sEH into their biologically less active corresponding dihydroxy metabolites (Zhang et al., 2014; Wang et al., 2018).

The role of epoxidized LC-PUFAs is well established in several biological processes, such as angiogenesis, inflammation, and tumor growth: In different animal models, it has been shown that sEH-inhibition has a positive influence on cardiovascular and liver abnormalities (Iyer et al., 2012), liver fibrosis and portal hypertension (Zhang et al., 2018), fatty liver (Yao et al., 2019) and non-alcoholic steatohepatitis (Wang et al., 2019). The EETs are known to affect blood pressure, inflammation, pain sensation, and regeneration (Arnold et al., 2010; Morisseau et al., 2014). However, a proangiogenic effect of 11,12-EET and 14,15-EET, the main EET regioisomers in mammals (Spector and Kim, 2015), has been described via the epidermal growth factor (EGF) and VEGF pathways (Zhang et al., 2014), which may explain the finding that EETs promote tumor growth. The n-6 AA-derived regioisomers 5,6-EET and 8,9-EET were found to increase cell proliferation and de novo vascularization (Yan et al., 2008), whereas 11,12-EET and 14,15-EET promote tumor angiogenesis through endothelial cell proliferation (Panigrahy et al., 2012; Zhang et al., 2013). An increase in 14,15-EET through sEH-inhibition led to increased tumor growth and metastasis through cell invasion in experimental studies (Panigrahy et al., 2012). In summary, through mechanisms of cell proliferation, de novo vascularization and endothelial proliferation several EET-regioisomers promote tumor growth and metastasis (Yan et al., 2008; Panigrahy et al., 2012; Zhang et al., 2013; Zhang et al., 2014). The sEH-inhibitory effect of sorafenib might thus carry clinically relevant consequences by increasing these pro-tumorigenic EET mediators.

In contrast, n-3 PUFA-derived regioisomers show anti-angiogenic effects both via the VEGF and FGF-2 pathway (Zhang et al., 2014). In a tumor mouse model, it was shown, that low dose sEH-inhibition led to an increase in n-3 DHA-derived 19,20-EDP and thereby reduced tumor angiogenesis and cell invasion and thus inhibition of tumor growth (Zhang et al., 2013). Furthermore, a protective effect in obesity and obesity-related comorbidities, such as fatty liver disease, of n-3 epoxy PUFA was found in animal models (López-Vicario et al., 2015). The beneficial effects of n-3 PUFA regarding to cancer and its complications are probably due to their anti-inflammatory and pro-resolution mediators (Freitas and Campos, 2019). In the context of abundant DHA, the sEH-inhibitory effect of sorafenib might thus lead to higher levels of 19,20-EDP, mediating anti-tumor effects.

Currently, n-6 PUFA are found in a ratio of approximately 20 times more than n-3 PUFA in the human diet (Harris, 2006); therefore humans have low n-3 PUFA tissue levels, and a shift of the competitive n-6 and n-3 PUFA metabolism towards n-6 PUFA derived lipid metabolites (Spector and Kim, 2015).

We therefore aimed to investigate levels of n-6 and n-3 PUFA in HCC patients, as well as n-6 PUFA- and n-3 PUFA-derived epoxide and corresponding dihydroxy compounds in HCC patients without and during sorafenib therapy. Based on the data of our pilot study (Leineweber et al., 2020), we hypothesized that sorafenib treatment, due to sEH-inhibitory and possibly CYP-modulating effects, might increase the presence of potentially tumor growth-suppressing DHA-derived EDPs, as well as of potentially tumor growth-promoting EETs.

Blood samples from a total of n = 43 HCC patients were analyzed in a paired fashion with blood taken without and undergoing 7–9 weeks sorafenib treatment. This is a sub-analysis of the well-characterized study population of the randomized controlled, multicenter phase II SORAMIC trial (Ricke et al., 2019). Patients from this study population, from which suitable amounts of blood samples were available for the analysis of fatty acids and their lipid metabolites before and during sorafenib treatment, were chosen for the analysis performed and presented here. The patients received sorafenib 200 mg twice a day for 1 week before increasing the dose to 400 mg twice a day. Based on disease progression and clinical condition, the sorafenib dose was escalated to 600–800 mg or reduced to 0–200 mg. The patients’ general characteristics are shown in Table 1.

We found significant differences in plasma fatty acid composition in patients with HCC without sorafenib compared to during sorafenib treatment. Relative levels of AA and DHA were significantly lower during sorafenib treatment. Furthermore, we were able to demonstrate significantly higher EET levels and a trend towards increased n-3 CYP metabolites especially 19,20-EDP in this study population with HCC receiving sorafenib treatment.

When taking into account the different levels of the precursor n-3 PUFAs EPA and DHA we were able to establish that EPA is metabolized by CYP enzymes to a similar extent as AA, while DHA utilization was higher, leading to significantly increased levels of the 19,20-metabolites derived from DHA during sorafenib treatment (Figures 2E, 5B). Given that previous data from mouse models show inhibition of tumor angiogenesis and reduced cell invasion by increasing 19,20-EDP (Zhang et al., 2013) and to dampen and alleviate inflammation in the liver (López-Vicario et al., 2015), this could be a beneficial effect of sorafenib that could be harnessed in HCC therapy by supplementing DHA.

Generally, a beneficial role of n-3 PUFAs to dampen development of HCC is described both in animal models (Lim et al., 2009; Weylandt et al., 2011; Inoue-Yamauchi et al., 2017) and human observation studies (Sawada et al., 2012; Gao et al., 2015; Koh et al., 2016). This and the disbalance of the n-6/n-3 ratio in the Western Diet (Harris, 2006) suggests that supplementation of n-3 PUFAs could balance the n-6/n-3 ratio and may reduce tumor progression in HCC patients, regardless of sorafenib treatment. With all the described effects of n-3 PUFAs as receptor agonists, modulators of molecular signalling pathways and inflammatory responses, and data indicating that n-3 PUFA increase the efficacy of chemotherapies and consequently the overall survival of cancer patients, n-3 PUFAs can thus be considered as pharmaceutical nutrients (Bougnoux et al., 2009; Chagas et al., 2017; Paixão et al., 2017). However, in the population studied here the n-3 PUFA baseline was not associated with the overall survival (Supplementary Figure S2).

Prior studies showed that dietary increase of baseline n-3 PUFA concentrations can enhance formation of n-3 PUFA-derived CYP epoxy-PUFA (Fischer et al., 2014; Sarparast et al., 2020; Weylandt et al., 2022). Higher levels of n-3 PUFA may thus potentially increase anti-tumor n-3 PUFA-derived epoxymetabolites as well as decrease pro-tumor n-6 PUFA-derived metabolites (Zhang et al., 2014). Interestingly, in this study we found lower levels of DHA in patients treated with sorafenib (Figure 3), further supporting the concept to increase DHA in the daily diet in order to increase also levels of 19,20-EDP in HCC patients treated with sorafenib.

Many classes of currently used drugs can block or modify pathways of lipid mediator formation. Particularly well-established are non-steroidal anti-inflammatory drugs inhibiting the cyclooxygenase (COX) enzymes as well as numerous clinically well-established substances that modify (induce, inhibit) CYP enzymes and thereby modify lipid mediator formation. In general, by using quantitative LC-MS/MS oxylipin analysis in the context of established pharmacotherapy (pharmacolipidomics) as shown in this paper we hope to identify oxylipins that might be used to stratify and possibly also modify and improve response to treatments: Experimental data indicate strong biological effects of specific lipid mediators, particularly with regard to inflammation-dampening oxylipins from n-3 PUFA (n-3 IDOs) (Weylandt et al., 2022) in contrast to often inflammation-promoting oxylipins from n-6 PUFA (n-6 IPOs). As concentrations of these can be modified by changes in fatty acids substrates, as well as established drugs such as sorafenib, there could be a rationale for targeted modifications of the n-6/n-3 PUFA ratio in the diet in the context of established pharmacotherapy to harness these effects.

In systemic HCC therapy, the combination of immune checkpoint inhibitors, and VEGF pathway inhibitors such as sorafenib could promote an immune-permissive environment, thereby enhancing the response to immune therapeutic approaches. Immunotherapy is effective in only approximately 1/3 of cancer patients, and targeting the TME to decrease tumor cell evasion is regarded as an opportunity to improve response to immunotherapy, as conversion of “cold” tumors to “hot” tumors with T cell infiltration is associated with a better response rate to cancer treatment (Bonaventura et al., 2019). TME-modifying properties of lipid mediator levels may therefore enhance antitumor effects by transforming the immune landscape. Aspirin dampens tissue inflammation via the metabolism of arachidonic acid in the COX enzymatic pathways and can reduce cellular growth in hepatocellular carcinoma (Tao et al., 2018; Refolo et al., 2020). Whether a CYP/sEH-dependent effect on lipid mediators—which could be modulated/enhanced by sorafenib as described here—could also play a role in the TME, remains a topic for future studies that are directly analyzing effects of n-3 IDO and n-6 IPO levels and formation, as well as immune cells in liver and liver tumor tissue.

In our data presented here, we were not able to discern an sEH inhibitory effect of sorafenib (Figure 4). Non-etheless we established significantly higher levels of CYP-derived epoxy and dihydroxy metabolites in patients undergoing sorafenib treatment. While previous results from one of us show that storage at −80°C should be sufficient to yield stable CYP derived oxylipin readings comparable to other oxylipins (Gladine et al., 2019; Koch et al., 2020), there might have been variations in the process of blood sampling and storage leading to changes in epoxy-PUFA levels. Another explanation would be that sorafenib may have more complex effects on PUFA-derived metabolites in humans, with increased formation of epoxy-PUFAs. We did not analyse expression of sEH directly, therefore there is a possibility of increased sEH expression, as described in an animal models of high fat diet induced liver disease (López-Vicario et al., 2015) which might compensate for an inhibitory effect of sorafenib. Interestingly, in our pilot study we found an increase of 8,9-EET, 11,12-EET and 14,15-EET levels in HCC patients treated with sorafenib (Leineweber et al., 2020) while we found a significant increase only of 5,6-EET and 8,9-EET levels here. We believe this might be due to the analytical limitations due to differences in sample taking and storage, and not a mechanistic difference in the effect observed. Indeed, we suggest to use the combined analysis of epoxides and dihydroxy compounds (as in Figures 2D–F) to assess epoxy metabolite formation. However future studies could better address this question by analyzing the samples under defined conditions and at different time intervals after blood drawing to measure the effects of these variations on epoxy- and dihydroxy-PUFA levels.

In this study, we investigated the effect of sorafenib treatment on PUFA formation and epoxy lipid mediator concentrations in peripheral blood plasma in a group of 43 HCC patients as a sub-analysis of the randomized, controlled, multicenter phase II SORAMIC study. We were able to demonstrate markedly increased epoxy plus dihydroxy PUFA concentrations in the peripheral blood of HCC patients undergoing sorafenib therapy. These results support previous findings that sorafenib treatment induces a change in epoxy-/dihydroxy-PUFA concentrations.

Given the anti-tumor effects described in experimental models for the n-3 PUFA-derived 19,20-EDP, these data further support the hypothesis that dietary n-3 PUFA supplementation in addition to sorafenib treatment could contribute anti-tumor effects due to n-3 epoxy-PUFA.