Hippo signaling is an emerging tumor
Hippo signaling is an emerging tumor suppressor pathway that plays key roles in normal physiology and tumorigenesis through the regulation of cellular proliferation and survival . In humans, YAP is over-expressed as a result of genomic amplification of the 11q22 locus in a wide spectrum of human cancer cell lines and primary tumors [8,9]. In addition, several lines of evidence suggest that extracellular ligands modulate the Hippo pathway through GPCR signaling . So it is very important to explore whether DHA induced cell growth inhibition and apoptosis of human prostate cancer MK-1775 receptor via YAP. As the results shown in Fig. 2, DHA induced YAP phosphorylation in androgen-independent prostate cancer cell lines PC3 and DU145 but not in androgen-dependent prostate cancer cell line LNCaP. To verify this, we further investigate whether DHA promotes LATS phosphorylation in PC3 and LNCaP. Not surprisingly, DHA phosphorylates and activates LATS in PC3 but not LNCaP, suggesting DHA inhibits LNCaP proliferation in a different way. Our previous study has shown that DHA inhibits the growth of hormone-dependent prostate cancer cells LNCaP via promoting the degradation of the androgen receptor. The reason for this discrepancy may be that the mechanism of DHA induces cell growth inhibition and apoptosis are various in cell lines of different species and backgrounds. Moreover, it is known that LATS1/2 inactivates YAP by phosphorylating its Ser127 and Ser381 residues, Ser127 phosphorylation mediates interaction with 14-3-3 proteins, which sequester YAP in the cytoplasm . On the other hand, Ser381 phosphorylation triggers successive phosphorylation on Ser384 by casein kinase-1 followed by SCFβ−TRCP E3 ubiquitin ligase-mediated degradation . Our data has shown that the protein levels of YAP and TAZ were unaffected by DHA stimulation, suggesting that DHA potently inactivate YAP by phosphorylating its Ser127 residue, thus inducing YAP transfer to cytoplasm. It has been reported that the pathway upstream of YAP phosphorylation is operative in a tissue or context-specific manner. LPA or S1P bound to their corresponding membrane GPCRs and act through Rho GTPases to activate YAP/TAZ . Consistently, another report showed that activation of PAR1 (a GPCR) that couple to G12/13 triggers the activation of Rho GTPase, which works through the actin cytoskeleton to inhibit LATS1/2 kinase, leading to eventual YAP/TAZ dephosphorylation, nuclear localization, and gene expression . However, the signaling mechanism linking Gαs-coupled GPCRs to LATS activation was not elucidated. A recent study showed that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate LATS kinases and YAP phosphorylation . These findings suggest that the activity of YAP can be positively or negatively modulated by a wide range of extracellular signals via GPCRs in a manner dependent on which G proteins is stimulated. As the results shown in Fig. 3, we found that DHA inactivates YAP by inducing phosphorylation dependent on FFAR1, FFAR4, Gαs and PKA. In addition, it is well known that SAV1 facilitates MST1/2 kinases to phosphorylate and activate LATS1/2, in turn, phosphorylate and inactivate the transcriptional coactivator YAP. However, previous studies showed that extracellular ligands bound to their corresponding membrane GPCRs and modulate YAP activity in LATS-dependent or LATS-independent manner [11,25]. The data in this paper clearly showed that DHA increases YAP phosphorylation dependent on the canonical Hippo pathway. We found that a reduction of YAP phosphorylation when the protein level of LATS1 and MST1 have been knockdown and DHA-induced YAP phosphorylation was reversed by both the LATS1 and MST1 siRNAs. Moreover, we can also found that both the phosphorylation of MST1 and LATS1 increased following DHA treatment, further proved DHA inactivates YAP through FFAR1 and FFAR4 in the canonical Hippo pathway-dependent manner (Fig. 3A–D). In conclusion, our findings provide a novel insight into how DHA modulates androgen-independent prostate cancer cells proliferation.