OA treatment also reversed LPA induced dpTAZ, consistent with an important role for a PP in LPA induced dephosphorylation of YAP and TAZ in OVCA433 cells. To determine which PP was involved, siRNAs against the catalytic subunits of PP1 and PP2 were used. LPA induced dpYAP was reversed by the PP1A but not the PP2A siRNA, suggesting that PP1A Enzastaurin Phase 3 is activated by LPA and YAP is likely to be a direct substrate Inhibitors,Modulators,Libraries of PP1A. The specificity of the siRNA down regulation of PP1A and PP2A is shown in Figure 5E. To determine whether PP1A is up or down stream of RhoA ROCK, we used the constitutively active form of RhoA. The ca RhoA was able to induce dpYAP in an OA sensitive manner, suggesting that PP1A was down stream of RhoA. The expression of transfected RhoA was confirmed using RhoA antibody.

LPA induced AREG secretion and EGFR dependent cell migration was LPA3 G13 RhoA ROCK PP1 dpYAP dependent The mechanisms by which YAP signaling affects cell mi gration has been only minimally studied. Since YAP is a transcriptional co activator and AREG, an EGFR lig and, has been identified as a YAP and TAZ Inhibitors,Modulators,Libraries target, we tested whether EGFR was involved in LPA induced cell migration in a YAP dependent manner. We found that AG1478, an EGFR selective inhibitor, did not inhibit LPA induced dpYAP, but did inhibit LPA stimulated cell migration, suggesting that an EGFR ligand may be a target of YAP. We tested AREG directly and showed that indeed, AREG induced an AG1478 sensitive cell migration. The involvement of EGFR and AREG was further supported by the actions of a second EGFR inhibitor, PD153035 on both pYAP and migration.

OVCA433 cells demonstrated a basal secretion Inhibitors,Modulators,Libraries of AREG, as measured by an increased AREG in conditioned medium over time. LPA stimulated AREG secretion above the basal level, correlated to the increase in AREG mRNA expression, which peaked at 8 hr. An siRNA against YAP reduced both basal and LPA induced AREG secretion from the OVCA433 cells. To confirm the signaling pathway, we tested the potential in volvement of several key molecules in AREG section. As shown in Figure 6B, c, down regulation of YAP and LPA3, but not LPA1, completely Inhibitors,Modulators,Libraries abolished LPA induced AREG se cretion. LPA induced AREG secretion was also sensitive Inhibitors,Modulators,Libraries to dn G13, dn RhoA, Y27632, and OA, but not PTX or dn Gq. consist ent with the LPA induced YAP signaling pathway.

In addition, LPA induced AREG secretion was actinomycin D and cyclohexamide sensitive, suggesting that both transcription and translation processes are in volved. LPA induced cell animal study migration has been extensively studied in EOC and other cancer cells. However, the current work is the first to show the involvement of YAP in LPA actions in EOC cells. Relatively short times are traditionally used for Transwell migration studies in EOC cells. Since YAP is a transcriptional co activator, we tested migration over a longer time.