In early a collaborative effort between the group that pione

In early 2017, a collaborative effort between the group that pioneered the above described evidence of platinum-induced PUFA chemotherapy resistance and a group that has contributed to much of the known molecular pharmacology of FFA4, revealed that the molecular target of 16:4(n-3), which affords chemotherapy resistance, is indeed FFA4 [89]. In a similar manner as that seen for 12-S-HHT/BLT2, it was shown that 16:4(n-3) agonizes FFA4 on the surface of F4/80+/CD11blow macrophages to modulate this effect. Interestingly and as may be expected, in clonal cell models, 16:4(n-3) activated both Gαq/11 as well as β-arrestin-2 linked signaling to both FFA4 and FFA1, demonstrating that it is an agonist at both long-chain fatty Cdk1/2 Inhibitor III receptors [89]. In a well-designed model utilizing conditioned media from mouse splenic cells treated with 16:4(n-3), GW9508, or TUG-891, it was shown that tumor xenografts that were markedly reduced in volume upon treatment with cisplatin, were insensitive to the effects of the platinum agent when it was co-administered with the splenic conditioned media (sCM), suggesting an important role for the FFA receptors in chemotherapy resistance in vivo [89]. Further studies using the selective antagonists of FFA4 and FFA1, AH7614 and GW1100, respectively, showed that 16:4(n-3)-induced chemoresistance was modulated by FFA4 and not FFA1, as tumor volume was significantly decreased by cisplatin/16:4(n-3) + AH7614 sCM but not by cisplatin/16:4(n-3) + GW1100 sCM [89]. These results were confirmed to be FFA4 mediated and specific to 16:4(n-3), as sCM derived from FFA4−/− mice was incapable of inducing 16:4(n-3) chemoresistance, while it had no effect on 12-S-HHT chemoresistance, which as noted previously, occurs via agonism of BLT2. While F4/80−/CD11blow, F4/80+/CD11bhigh, and F4/80−/CD11bhigh macrophage subpopulations did not appear to express FFA4, the effects of 16:4(n-3) were shown to occur via agonism of FFA4 localized to F4/80+/CD11blow splenic macrophages, which expressed FFA4 and FFA1, in addition to the short-chain fatty acid receptors FFA2 and FFA3 [89]. Within F4/80+/CD11blow cells, agonism of FFA4 by 16:4(n-3) was shown to cause a rapid phosphorylation and increases in activity of cytosolic phospholipase A2 (cPLA2), which was blocked by the FFA4 antagonist AH7614, and was not reproduced in other macrophages [89]. Mechanistically, FFA4-mediated cPLA2 activity facilitated the generation of a 24:1 unsaturated lysophosphatidylcholine (LPC24:1), which directly modulates chemoresistance [88], [89] (Fig. 3). The specific role of this FFA4-cPLA2-LPC24:1 axis was confirmed using sCM from FFA4−/− mice, pharmacological inhibition of cPLA2, and direct administration of LPC24:1 [89]. Finally, the research team demonstrated that human CD163+ splenocytes have heightened expression of FFA4, similar to that seen with orthologous murine F4/80+ cells, and that humans subjected to four hours of platinum-based chemotherapy had significantly higher levels of plasma LPC24:1 compared to those that had undergone non-platinum based therapies, suggesting that this mechanism is directly translatable to human chemoresistance.
Concluding remarks Since the first study on the role of FFA4 in cancer cells was published by Wu and colleagues in 2013 [51], many other reports have been published, establishing a linkage to either promotion or inhibition of proliferation and migration of prostate, colorectal, lung, pancreatic, skin, and breast cancers, as well as chemoresistance. As described here, many of these cancer cells express both long-chain FFA receptors. Given that many of the initial studies made use of GW9508, an agonist with more selectively for FFA1, but which also agonizes FFA4, interpretation of the effects in these cells is somewhat cloudy. The utilization of siRNA/shRNA-based approaches does shed light on the role of FFA4 in these studies, however, since some of the findings do not report the efficacy of FFA4-knockdown, again, it is difficult to pinpoint a definitive role for FFA4 in the case that FFA1 is present and non-selective pharmacological agents are used. Since many of the studies also present evidence that the two FFA receptors have opposing actions on proliferation, migration, and or invasion, it is conceivable that loss of activity of one receptor via knockdown approaches may dysregulate the opposing balance, particularly when non-selective agonists are used to compare effects in control cells. Further work will be needed using more selective agonists (e.g., TUG-891, cpdA) as well as antagonists, such as AH7614, whose activity was first noted in 2014, to more clearly define the role for FFA4 in cancers. While the studies performed thus far delineate involvement of FFA4 in many of these cancers, the detailed intracellular cascades at play in each cancer type, and likely even each cell type, will also require further study in order to understand the mechanisms of cell signaling that link FFA4 to its anti- or pro- cancerous effects in each given tissue. Since FFA4 is agonized by a variety of medium-to-long-chain fatty acids, including MUFA, omega-3, -6, and -9 PUFA, as well as saturated fats, all of which have historically been described either as “good” or “bad” fats, it is difficult at this point to link the receptor to a definitive benefit or risk towards cancer signaling. Given the possibility and context of these fats inducing agonist-directed functional selectivity via FFA4, further study will be needed to conclusively expound the role of the receptor in these and other cancers, and to elucidate the cellular biology employed by receptor activation.