In stark contrast to this study Zhang and colleagues
In stark contrast to this study, Zhang and colleagues demonstrated that FFA4, along with FFA1, play roles in suppressing cell proliferation and promoting apoptosis in CRC mao inhibitor treated with omega-3 PUFA . In this study design, in vivo CRC was induced in mice upon treatment with the genotoxic colon carcinogen azoxymethane (AOM), in combination with pro-inflammatory dextran sulfate sodium (DSS), and mice were fed control diets (AIN93) or the same diet supplemented with omega-3 PUFA that included 33% EPA and 23% DHA for 11 weeks. Given this paradigm, AOM/DSS treatment induced tumors in 93% of mice fed control diets and 55% of mice fed the omega-3 supplemented diet , although as seen in other studies using similar fats , the later animals gained more weight . The tumors seen in omega-3 supplemented diet animals were smaller (12.4 versus 25.8 mm) and fewer in number (1.8 versus 4.2) compared to control-diet fed animals . To investigate the role of FFA4 in this model, the investigators utilized LOVO and HT-29 CRC cell lines, the latter of which had previously been shown to express over two-fold higher FFA4 as described above . Both DHA and EPA exhibited dose- and time-dependent anti-proliferative effects in both cell lines, and moreover, both omega-3 PUFAs elicited significantly higher levels of apoptosis . The transcriptional regulators within the HIPPO-YAP/TAZ pathway play critical roles in cell proliferation and organ growth and development. Dysregulation of HIPPO pathway signals can also elicit initiation, growth, and metastasis of tumors, particularly CRCs , , . In its unphosphorylated state, YAP is localized to the nucleus where it can regulate transcription to drive proliferation and survival as well as aberrant cell growth; while phosphorylation on Ser127 facilitates its redistribution to the cytosol, where it remains sequestered and can be proteosomally degraded. GPCRs are known to modulate the HIPPO-YAP/TAZ system via Gαs/cAMP/PKA-dependent phosphorylation of upstream LATS1/2, leading to phosphorylation of YAP/TAZ and inhibition of its nuclear translocation, or alternatively, via signaling to F-actin via Rho GTPases that modulates opposing effects ,  (Fig. 2). Based on this evidence, the effects of omega-3 PUFA on FFA4-linked HIPPO signaling was investigated and it was found that both DHA and EPA facilitate the dose- and time- dependent phosphorylation of YAP in both LOVO and HT-29 CRC cells . Moreover, PUFA treatment modulated cytosolic retention, rather than nuclear translocation, of YAP/TAZ in both CRC cell lines. Further study on this pathway revealed that indeed, the anti-proliferative and apoptotic effects of DHA and EPA were mediated via YAP through this canonical HIPPO pathway. Importantly, this study found that the effects of DHA and EPA were modulated via both FFA1 and FFA4 in these CRC cell lines, and that the PKA inhibitor H-89 abrogates these effects, suggesting that they are mediated via FFA1/FFA4 coupling to the Gαs/cAMP/PKA cascade . Finally, the research group investigated the effects of omega-3 enriched diets on YAP/TAZ in vivo using the AOM/DSS CRC model and found that YAP/TAZ were increased in CRC tissue compared to normal tissue, and that YAP and TAZ were significantly reduced in CRC tissues from animals fed omega-3 enriched diets. As seen in CRC cell lines, phosphorylated YAP was also increased in tumors of animals fed omega-3 enriched diets and these animals also expressed lower levels of YAP targeted genes related to proliferation . These two studies are the only ones that delineate a role for FFA4 in CRC and offer markedly contrasting views on the role of the receptor in these cancers. Interestingly, the work of Wu and colleagues  did not detect expression of FFA1 in human CRC tissues while the results of Zhang did . Further, the results from the former study showed no expression of FFA1 in either CRC cell line used – HCT116 and SW480; while those from the latter show that LOVO and HT-29 CRC cell lines do indeed express FFA1. Hence, there are mixed observations on the expression and role of either long-chain PUFA receptor that can confound the interpretations of data that are derived, particularly given that non-selective FFA1/FFA4 agonists such as EPA, DHA, and GW9508 are used in these studies. The use of selective agonists such as TUG-891, or inclusion of the recently reported FFA4 antagonist AH7614  in future studies should more clearly delineate the role of FFA4 in CRCs. This is especially important given the nature of the described coupling of FFA4 and FFA1 to Gαs rather than the predominately described Gαq/11 effects, raising questions regarding biased-signaling or functional selectivity of one or both receptors in given cell types. The other possible variable in the context of CRCs is the known expression of the FFA4-L isoform in these cells and the possible role that it plays in concert or opposition to those of the shorter isoform. A final point regarding the differences seen in the CRC studies concerns the temporal differences in study design and measure of the outcomes. Much of the work of Zhang and colleagues was done under the context of longer term stimulations with agonists (e.g., 6 h or more), compared to those done by Wu and colleagues that assessed more rapid measures that occurred on the order of minutes to a few hours. Nonetheless, in vivo tumor progression was markedly progressed by FFA4 in one study, and markedly reduced by FFA4 in the other, so much more work remains to be done to clearly elucidate the cell biology involved.