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, 11 (1), 31

The Role of LPA and YAP Signaling in Long-Term Migration of Human Ovarian Cancer Cells

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The Role of LPA and YAP Signaling in Long-Term Migration of Human Ovarian Cancer Cells

Hui Cai et al. Cell Commun Signal.

Erratum in

  • Cell Commun Signal. 2013;11:92

Abstract

Background: The Hippo-YAP signaling pathway is altered and implicated as oncogenic in many human cancers. However, extracellular signals that regulate the mammalian Hippo pathway have remained elusive until very recently when it was shown that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) ligands including lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P). LPA inhibits Lats kinase activity in HEK293 cells, but the potential involvement of a protein phosphatase was not investigated. The extracellular regulators of YAP dephosphorylation (dpYAP) and nuclear translocation in epithelial ovarian cancer (EOC) are essentially unknown.

Results: We showed here that LPA dose- and time-dependently induced dpYAP in human EOC cell lines OVCA433, OVCAR5, CAOV3, and Monty-1, accompanied by increased YAP nuclear translocation. YAP was involved in LPA-induced migration and invasion of EOC cells and LPA3 was a major LPA receptor mediating the migratory effect. We demonstrated that G13, but not or to a lesser extent G12, Gi or Gq, was necessary for LPA-induced dpYAP and its nuclear translocation and that RhoA-ROCK, but not RhoB, RhoC, Rac1, cdc42, PI3K, ERK, or AKT, were required for the LPA-dpYAP effect. In contrast to results in HEK293 cells, LPA did not inhibit Mst and Lats kinase in OVCA433 EOC cells. Instead, protein phosphatase 1A (PP1A) acted down-stream of RhoA in LPA-induction of dpYAP. In addition, we identified that amphiregulin (AREG), a down-stream target of YAP which activated EGF receptors (EGFR), mediated an LPA-stimulated and EGFR-dependent long-term (16 hr) cell migration. This process was transcription- and translation-dependent and was distinct from a transcription- and YAP-independent short-term (4 hr) cell migration. EOC tissues had reduced pYAP levels compared to normal and benign ovarian tissues, implying the involvement of dpYAP in EOC pathogenesis, as well as its potential marker and/or target values.

Conclusions: A novel LPA-LPA3-G13-RhoA-ROCK-PP1A-dpYAP-AREG-EGFR signaling pathway was linked to LPA-induced migration of EOC cells. Reduced pYAP levels were demonstrated in human EOC tumors as compared to both normal ovarian tissues and benign gynecologic masses. Our findings support that YAP is a potential marker and target for developing novel therapeutic strategies against EOC.

Figures

Figure 1
Figure 1
LPA-induced dpYAP and YAP nuclear translocation in EOC cells. OVCA433 (A) and OVCAR5 (B) cells were starved for 16 hr, then treated with LPA (10 μM) for different times or with different concentrations of LPA for 2 hr. Western blots analyses and quantification methods were described in Materials and Methods. Representative results are shown from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. C, LPA (10 μM, 2 hr)-induced dpYAP was tested in two more EOC cell lines. D and E, LPA-induced YAP nuclear translocation is shown in OVCA433 and OVCAR5 cells. Green: YAP; red: DAPI. F, LPA (10 μM) time-dependently induced de-phosphorylation of TAZ.
Figure 2
Figure 2
LPA-induced migration and invasion of EOC cells was reduced by YAP-siRNA. A, Reduced YAP expression by YAP siRNA in OVCA433 and OVCAR5 cells detected by Western blot. B and C, The effect of down-regulation of YAP on cell migration and invasion induced by LPA (10 μM) in OVCA433 and OVCAR5 cells (conducted 48 hr-post siRNA treatment). The results are from three independent experiments. **P < 0.01.
Figure 3
Figure 3
LPA-induced dp-YAP and YAP nuclear translocation were dependent on Rho/ROCK, but independent of PI3K, MEK, p38, and AKT. After starved from FBS for 16 hr, cells were pretreated with different inhibitors, SB203580 (10 μM), LY294002 (10 μM), Ki16425 (10 μM), MK2203 (1 μM), Y27632 (10 μM), PD98059 (30 μM) for 1 hr, and C3 (1 μg/mL) for 2 hr prior to stimulation with LPA (10 μM, 2 hr). pYAP expression in OVCA433 (A) and OVCAR5 (B) cells was analyzed by Western blot. C, OVCA433 cells were treated as described in A. Cells were fixed and stained for YAP (green). Cell nuclei were stained with DAPI (red). Representative results are shown.
Figure 4
Figure 4
LPA3, but not or to lesser extent LPA1, LPA2, and LPA4, mediated the LPA-dpYAP effect. A, OVCA433 (a) and OVCAR5 (c) cells were starved and pretreated with Ki16425 (10 μM) for 1 hr prior to treatment with LPA (10 μM, 2 hr). pYAP was analyzed by Western blot. (b) The effect of Ki16425 on LPA (10 μM, 2 hr)-induced YAP nuclear translocation in OVCA433 cells. Green: YAP; red: DAPI. Representative results are shown. B, (a) The mRNA levels of LPA receptors after siRNA-treatment in OVCAR433 cells were determined by quantitative real-time PCR. Normalized expression values are given as percentage of control siRNA treated samples (means ± SD of three independent experiments). ***P < 0.001. (b) LPA (10 μM, 2 hr)-induced dpYAP effects were determined in LPA receptor specific siRNA-treated cells (48 hr post-transfection). (c) Quantitation of Western blots from (b) presented as fold decrease of pYAP after LPA stimulation compared to unstimulated controls. The data are means ± SD from three independent experiments. *P < 0.05. C, D and E, Cells were pretreated with PTX (100 ng/mL, 16 hr) or transfected with different dn plasmids for 48 hr, starved and then treated with LPA (10 μM, 2 hr). Cell lysates were analyzed by Western blot. Representative results are shown.
Figure 5
Figure 5
PP1A was involved in LPA-induced dpYAP and cell migration. A, Starved OVCA433 cells were treated with LPA (10 μM) for different times, and pMst1/2 and pLats were analyzed by Western blot. B, Starved OVCA433 and OVCAR5 cells were pretreated with OA (100 nM, 1 hr), followed by LPA (10 μM, 2 hr). pYAP and pTAZ were analyzed by Western blot. C, OVCA433 cells were treated as described in (B) and the effect of OA on cell migration was tested. *** P < 0.001. D, OVCA433 cells were transfected with control, PP1A, or PP2A siRNAs for 48 hr. The cells were starved for 16 hr and treated with or without LPA (10 μM, 2 hr). Cell lysates were analyzed by Western blot for pYAP. E, Specific down-regulation of PP1A or PP2A proteins. F, OVCA433 cells were transfected with the vector or the ca-RhoA plasmid, and then treated with or without OA (100 nM, 4 hr). RhoA expression was examined in cell lysates by Western blot analyses.
Figure 6
Figure 6
YAP-dependent amphiregulin (AREG) production/secretion mediated EGFR-dependent LPA-induced cell migration. A, The effects of AG1478 (1 μM, 1 hr pretreatment) and PD153035 (4 μM, 1 hr pretreatment) on LPA-dpYAP (a), or cell migration (b); AREG (100 pg/mL) induced AG1478 (1 μM, 1 hr)-sensitive migration of OVCA433 cells (c). *P < 0.05. B, Basal and LPA (10 μM)-induced AREG in conditioned media (CM) of OVCA433 cells (a). mRNA level of AREG was increased by LPA (b). LPA (10 μM, 8 hr)-induced AREG in CM was YAP- and LPA3-, but not LPA1-dependent (c). LPA-induced AREG production/secretion was blocked by dn-G13, dn-RhoA, but not PTX (100 ng/mL, 16 hr) or dn-Gq (d and e). C, LPA-induced short-term migration (4 hr) was not dependent on YAP or transcription (ActD, 1 μg/mL, 1 hr pretreatment), but was sensitive to PTX (100 ng/mL, 16 hr), LY294002 (10 μM, 1 hr pretreatment) and dn-Rac1 transfection (a). LPA-induced long-term cell migration was sensitive to ActD and CHX treatment (20 μg/mL, 1 hr pretreatment), as well as PTX and LY294002 (b). Data are from three independent experiments (means ± SD). *P < 0.05, **P < 0.01. D, Summary the LPA-YAP signaling pathway revealed in this work (OVCA433 cell line, left panel) and the previous work [1] (HEK293A cell line, right panel).
Figure 7
Figure 7
pYAP expression was low in EOC tissues when compared to both normal and benign ovarian tissues. A, Immunofluorescence staining of total YAP (green) in normal and EOC tissues showed that YAP was mainly cytosolic and nuclear (blue) in normal and EOC tissues, respectively (a). Different locations for total YAP in normal and EOC tissues were confirmed by IHC (b). B, EOC tissues express lower levels of pYAP than normal and benign tissues. The top row shows the negative controls of IHC when the first antibody was not added. Representative pictures are shown (a). Summary of the quantified IHC results from normal ovary (n = 8), benign ovarian tissues (n = 10), and EOC tissues (n = 27) (b).

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