Discussion
Accumulating evidence suggests that DHA can decrease the viability of cultured cancer cells and inhibit growth, invasion, and metastasis of malignant tumors in vivo, mechanistically associated with cell proliferation inhibition, apoptotic or non-apoptotic cell death induction, and angiogenesis suppression, etc [32], [33], [34]. A better understating of the mechanisms involved in the anticancer activity of DHA would promote its clinical utilization since this knowledge is required to establish mechanism-based biomarkers potentially valuable for future clinical trials. The data presented herein indicate that DHA could reduce the expression of PD-L1 in cultured A549, LLC, HepG2, and SMMC-7721 cancer cells, as well as LLC tumor xenograft. The inhibition of PD-L1 expression by DHA was followed by disruption of PD-L1/PD-1 interaction and increase of T cell activity reflected by elevated levels of Granzyme B and enhanced DHA-induced death. These improved immune responses by DHA, in turn, contributed to the significant reduction of tumor growth. The findings of the present study uncovered a novel mechanism underlying the anticancer activity of DHA. The changes of PD-L1-related immune response induced by DHA can be used as biomarkers for future clinical trials.
The expression of PD-L1 is regulated by various complex processes, including gene transcription, post-translational modifications, and exosomal transport [35]. In our study, DHA treatment did not change the PD-L1 mRNA in all cell lines tested, suggesting that the transcriptional mechanism was not involved in the down-regulation of PD-L1 by DHA. Post-translational modifications, mainly glycosylation, play a vital role in regulating the expression of PD-L1. Glycosylation can stabilize PD-L1, and targeting glycosylation of PD-L1 has been shown to promote the degradation of PD-L1 and inhibit its interaction with PD-1, leading to reversing PD-L1-mediated immune suppression [36]. Inhibition of PD-L1 glycosylation is supposed to change its molecular weight, which can be measured by western blot. We carried out a time-course experiment (12h and 24h) to analyze the changes in the expression and molecular weight of PD-L1 in response to DHA in several cancer cells and did not find a significant change in the molecular weight of PD-L1 compared with the untreated control, so we ruled out the involvement of glycosylation modification in the down-regulation of PD-L1 by DHA. Protein degradation is mainly mediated by the ubiquitin-proteasome and autophagy-lysosome pathway in mammalian cells [26,37,38]. The role of these two degradation systems was examined, and the data showed that blockage of the ubiquitin-proteasome pathway but not autophagy pathway significantly rescued the degradation of PD-L1 by DHA, supporting an essential role of the ubiquitin-proteasome pathway in DHA-mediated degradation of PD-L1.
PD-L1 is generally regulated by the Ub/proteasome pathway through E3 ligases [5,37,39,40] and deubiquitinase [41]. As a deubiquitinating enzyme, CSN5 is considered a prognostic marker for various cancers, and it can deubiquitinate IκBa, Snail, and PD-L1, thereby promoting tumor progression migration [41], [42], [43]. We found that DHA reduced the expression level of CSN5 in LLC cells and Lewis xenograft tumor tissues (Figure. 7A and B), which is consistent with the increased ubiquitination modification in DHA-treated cells (Figure. 6E and F), indicating the crucial role of CSN5 inhibition in DHA-mediated PD-L1 ubiquitin degradation. Palmitoylation of proteins is a post-translational process that can regulate protein transport, localization, turnover, and signal transduction [44]. Recent studies have found that PD-L1 palmitoylation can be blocked by FASN pharmacological inhibition [30], and the reduction of palmitoylation will further increase the Ub-dependent degradation of PD-L1 [29]. As a member of the ω-3 PUFA family, DHA was reported to inhibit FASN, affecting the balance between energy intake and consumption in cancer cells [45]. Therefore, we speculated that the decrease of FASN by DHA could inhibit the palmitoylation of PD-L1, which further promoted the Ub-dependent degradation of PD-L1. Indeed, the essential palmitoyltransferase DHHC5 was reduced by DHA (Figure. 7D), which supported our hypothesis. These findings provided mechanistic interpretation for DHA-induced PD-L1 degradation.
A bioavailability study has shown that the average basal concentration of DHA (without DHA supplementation) in human plasma is around 32.41 ± 14.07 µg/mL (98.66 ± 42.83 μM) [46]. After administration of fish oil or krill oil, the average plasma DHA level can reach almost 60 μg/mL (about 182 μM) [47]. The representative concentration of DHA used in the cell culture study was 150 μM, which is, therefore, an achievable concentration in vivo. The dose of DHA (2g/kg) used in the animal experiment was determined based on previous studies [48,49], which is comparable to the dose used in previous human studies after calculation using the conversion tables between species [50]. Collectively, the concentrations we used in the present study are relevant for human translation. In addition, in our previous study, we also investigated the effect of other types of fatty acids besides DHA on PD-L1 expression in cancer cells (Figure S5). Interestingly, we found that only DHA of the ω-3 PUFA family seemed to significantly reduce the expression of PD-L1, this phenomenon may be worth further exploration.
In summary, ω-3 PUFA DHA at clinically relevant concentrations is able to inhibit the expression of PD-L1 in cancer cells by accelerating its proteasome degradation, thereby exerting the anti-tumor activity through its immunomodulatory function (Figure. 8). The findings of the present study implicates that DHA holds the potential to be used as an immune-enhancer for cancer treatment and prevention.
