Geranylgeranylation signals to the Hippo pathway for breast cancer cell proliferation and migration

Abstract

Protein geranylgeranylation (GGylation) is an important biochemical process for many cellular signaling molecules. Previous studies have shown that GGylation is essential for cell survival in many types of cancer. However, the molecular mechanism mediating the cell survival effect remains elusive. In this report, we show that the Hippo pathway mediates GGylation-dependent cell proliferation and migration in breast cancer cells. Blockade of GGylation enhanced phosphorylation of Mst1/2 and Lats1, and inhibited YAP and TAZ activity and the Hippo-YAP/TAZ pathway-dependent transcription. The effect of GGylation blockade on inhibition of breast cancer cell proliferation and migration is dependent on the Hippo-YAP/TAZ signaling, in which YAP appears to regulate cell proliferation and TAZ to regulate cell migration. Furthermore, GGylation-dependent cell proliferation is correlated with the activity of YAP/TAZ in breast cancer cells. Finally, Gγ and RhoA are the GGylated proteins that may transduce GGylation signals to the Hippo-YAP/TAZ pathway. Taken together, our studies have demonstrated that the Hippo-YAP/TAZ pathway is essential for GGylation-dependent cancer cell proliferation and migration.

Introduction

Protein prenylations, including farnesylation and geranylgeranylation (GGylation), are important biochemical modifications for the function of many signaling proteins.1,2 These lipid modification processes, catalyzed by farnesyltransferase or geranylgeranyltransferase (GGTase), function in membrane translocation of signal proteins, particularly small GTPases, and are directly involved in the regulation of tumor genesis and progression.3, 4, 5 Ras GTPases, which are mutated in many solid tumors, require prenylation for their malignant transforming activity.6 Thus, protein prenylation is an essential biochemical process for tumor cell survival, growth, proliferation, migration and metastasis.7

Farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGPP), the two substrates for protein prenylation, are synthesized by mevalonate pathway.8 Statins, the inhibitors of 3-hydroxyl-3-methylglutaryl-CoA reductase that is a key enzyme in the mevalonate pathway, have been used to pinpoint prenylation signaling by inhibiting biosynthesis of farnesyl pyrophosphate and GGPP.9 Early studies found that mevalonate is essential for the proliferation of breast cancer cells.10 Inhibition of the mevalonate pathway in breast cancer cells by lovastatin induced expression of cell cycle inhibitors p21 and p27, and caused arrest of cell cycle at the G1 phase.11 Rescue experiments indicated that inhibition of biosynthesis of GGPP, but not farnesyl pyrophosphate or cholesterol, by statins induced apoptosis in breast cancer cells.12,13 Consistent with the GGPP rescue studies, inhibition of GGylation by GGTase inhibitors (GGTIs) also resulted in apoptosis and G1 arrest in lung cancer cells by upregulating p21 and p27, and inducing hypophosphorylation of RB,14 and produced a similar inhibitory effect on breast cancer cell growth to that of lovastatin,9 confirming that inhibition of GGylation is the cause for induction of apoptosis in cancer cells.

Many studies now have shown that statins and GGTIs induce apoptosis in cancer cells.14, 15, 16, 17 Blockade of GGylation by GGTIs also inhibits tumor growth in xenograft mice.14,17, 18, 19 Knockout of GGTase I β-subunit significantly inhibited lung tumor formation.18 Interestingly, estrogen receptor-negative breast cancer cells were more sensitive to inhibition of GGylation than estrogen receptor-positive cells.20, 21, 22 These studies suggest a GGylation-dependent cell survival signaling pathway in breast cancer cells that may be associated with tumor progression. However, the molecular mechanism underlying the susceptibility of breast cancer cells to GGylation inhibition-induced apoptosis and the signaling context of GGylation-dependent cell survival are still poorly understood.

In this report, we have demonstrated that the Hippo pathway mediates GGylation-dependent breast cancer cell proliferation and migration signaling. In the Hippo pathway, the YAP signaling appears to regulate GGylation-dependent proliferation, while the TAZ signaling appears to regulate GGylation-dependent migration. Gβγ and RhoA may be the direct GGylation effectors for signaling to the Hippo pathway. These findings provide a new insight into the role of GGylation in tumor genesis and progression. In addition, we have also shown that susceptibility to statin- or GGTI-induced cytotoxicity is correlated with the activity of the Hippo pathway in breast cancer cells. Thus, the activity of the Hippo pathway can be used as an indicator for prediction of the response of breast cancer patients in GGylation-targeted therapy.

Results

Inhibition of GGylation reduces proliferation and migration of breast cancer cells

To determine the cellular function of GGylation in breast cancer cells, MDA-MB-231 cells were treated with the GGTase I inhibitor GGTI-298 and the 3-hydroxyl-3-methylglutaryl-CoA reductase inhibitor atorvastatin, and the effects on cell proliferation and migration were examined. As shown in Figure 1a and Supplementary Figure S1, both atorvastatin and GGTI-298 significantly inhibited the proliferation of the cells as determined by either the thiazolyl blue tetrazolium bromide (MTT) or the cell counting assay. The effect of atorvastatin on proliferation is caused by inhibition of biosynthesis of GGPP, as the addition of geranylgeraniol (GGOH) in the culture medium rescued the inhibition. As expected, the addition of farnesol (FOH) did not rescue the inhibition, indicating that farnesylation is not involved in the effect. The rescue effect of coenzyme Q or squalene on atorvastatin-induced inhibition of cell proliferation was also examined and no significant effect was observed (data not shown). We noticed that treatment with atorvastatin had a much stronger inhibitory effect than GGTI-298 (Figure 1a). This difference may be due to the activity of GGTase II (Rab GGTase), which is not affected by GGTI-298. Similar to the effect on proliferation, migration of MDA-MB-231 cells was severely impaired by treatment with GGTI-298 or atorvastatin as determined by either the wound healing or the Transwell assay (Figures 1b and c). The inhibitory effect of atorvastatin on migration was rescued by the addition of GGOH, but not FOH, suggesting that GGylation is also required for cell migration of MDA-MB-231 cells. Taken together, these results demonstrated that GGylation has an important role in both proliferation and migration of MDA-MB-231 cells.

Figure 1
figure1

Inhibition of GGylation impairs proliferation and migration of breast cancer MDA-MB-231 cells. (a) Cells were seeded and incubated for 24 h, and then treated with DMSO, GGTI-298 (10 μM, the same hereafter), atorvastatin (Atv) (10 μM, the same hereafter), Atv plus FOH (10 μM, the same hereafter) or Atv plus GGOH (10 μM, the same hereafter) for 48 h. Cell proliferation was determined by MTT assay. Proliferation was calculated as the percentage versus DMSO. (b) The effect of treatment on cell migration was determined by the ability of the cells to migrate to the gap area. The wound closure was calculated as the percentage of wound area change after 14 h. (c) Migration of MDA-MB-231 treated with DMSO, GGTI-298, Atv, Atv plus FOH or Atv plus GGOH was assessed by transwell cell migration assay. Data are representative of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 (t-test); mean±s.e.m.

Inhibition of GGylation impairs the Hippo-YAP/TAZ signaling

To identify the protein effectors and intracellular signaling pathways that mediate the effect of GGylation on breast cancer cell proliferation and migration, the gene expression profiles of MDA-MB-231 cells upon treated with dimethyl sulfoxide (DMSO) (the solvent control), atorvastatin or atorvastatin plus GGOH were determined using the Affymetrix GeneChip microarray assay. The GGylation-associated gene was selected by two criteria: (a) the one that had at least twofold changes in expression upon atorvastatin treatment; and (b) the one whose change in expression induced by atorvastatin was rescued by GGOH to <1.5-fold of the control. Expression of 3030 genes (about 14% of total genes) was upregulated and 1304 genes (about 6% of total genes) downregulated by atorvastatin. There were 620 genes among the 3030 atorvastatin-upregulated genes (about 20%) whose expression was rescued by GGOH (associated with GGylation), and 789 genes among the 1304 atorvastatin-downregulated genes (about 61%) whose expression was rescued by GGOH (associated with GGylation).

Among the atorvastatin-downregulated GGylation-associated genes, there are several genes, such as CYR61, ANKRD1, BIRC5 and CTGF, are known target genes for the Hippo-YAP/TAZ pathway.23 This raises a connection of GGylation signaling to the Hippo-YAP/TAZ signaling. Thus, we aligned the 789 atorvastatin-downregulated GGylation-associated (GGOH-rescued) genes with the YAP- or TAZ-upregulated (>2-fold) genes detected in MCF10A cells.24 As shown in Figure 2a, among the 789 genes, as many as 327 genes (41%) are upregulated by TAZ, 242 genes (31%) upregulated by YAP and 198 genes (25%) upregulated by both TAZ and YAP. Further, we aligned the top 100 atorvastatin-downregulated GGylation-associated genes with the YAP- or TAZ-upregulated genes and found that 60 of the 100 genes are upregulated by TAZ, 68 of the 100 genes upregulated by YAP and 55 of the 100 genes upregulated by both TAZ and YAP. The data indicate that the Hippo-YAP/TAZ pathway is a major pathway regulated by GGylation signaling.

Figure 2
figure2

Inhibition of GGylation activates the Hippo signaling and suppresses YAP/TAZ activity. (a) A large portion of the atorvastatin (Atv)-downregulated GGylation-associated genes are the YAP/TAZ target genes. (Top panel) The alignment of the total 789 Atv-downregulated GGylation-associated genes with the YAP- or TAZ-upregulated genes; (bottom panel) the alignment of the top 100 Atv-downregulated GGylation-associated genes with the YAP- or TAZ-upregulated genes. (b) The mRNA level of CTGF and CYR61 was measured by real-time PCR. MDA-MB-231 was treated with DMSO, GGTI-298, Atv, Atv plus FOH or Atv plus GGOH for 24 h. (c) GGylation blockade enhanced the phosphorylation of Mst1/Mst2 and Lats1. MDA-MB-231 cells were treated with DMSO, GGTI-298, Atv, Atv plus FOH or Atv plus GGOH for 24 h. Mst1, Mst2 or Lats1 was immunoprecipitated and detected by immunoblot. (d) GGylation blockade induces phosphorylation of YAP and reduces TAZ amount. MDA-MB-231 cells were treated with DMSO, GGTI-298, Atv, Atv plus FOH or Atv plus GGOH for 24 h. TAZ was immunoprecipitated and detected by western blotting. (e) Inhibition of GGylation blocked YAP nuclear localization. MDA-MB-231 cells were treated with DMSO, GGTI-298, Atv, Atv plus FOH or Atv plus GGOH for 24 h. Scale bar, 20 μm. The graph shows the percentage of total cells with nuclear YAP. Data are representative of at least three independent experiments. **P<0.01 and ***P<0.001 (t-test); mean±s.e.m.

We then selected two known target genes of the Hippo-YAP/TAZ pathway, CYR61 and CTGF, from the top atorvastatin-downregulated GGylation-associated genes as readouts to determine the effect of GGylation inhibition on the Hippo-YAP/TAZ signaling. We confirmed the GGylation-associated expression of CTGF and CYR61 in MDA-MB-231 cells with quantitative real-time–PCR (qRT–PCR) (Figure 2b). Further, we examined the Hippo pathway in response to inhibition of GGylation. The phosphorylation of the Hippo pathway proteins Mst1/2 and Lats1 were detected using the immunoprecipitation assay to eliminate nonspecific phosphorylated proteins in the lysates that crossreact with anti-phospho-Mst1/2 or anti-phospho-Lats1 antibodies. As shown in Figure 2c, phosphorylation of Mst1/2 was enhanced by the GGTI-298 treatment. Phosphorylation of Lats1 was stimulated markedly by GGTI-298 and atorvastatin treatment and diminished by adding GGOH, suggesting that inhibition of GGylation in MDA-MB-231 cells activates the Hippo pathway. Consistent with the activation of Hippo signaling, GGTI-298 or atorvastatin treatments significantly reduced the amount of TAZ and enhanced the YAP phosphorylation at Ser127 (Figure 2d), which is a Lats1 phosphorylation site that inhibits YAP nuclear localization.23 As expected, GGTI-298 or atorvastatin treatment blocked translocation of YAP to the nuclei, and the inhibitory effect by atorvastatin was rescued by GGOH, but not FOH (Figure 2e). These results indicate that inhibition of GGylation stimulates the Hippo signaling and inhibits YAP/TAZ activity.

We further examined whether GGylation signaling is essential for the activation of YAP and TAZ, the effectors of the Hippo pathway. Lysophosphatidic acid (LPA)-mediated G-protein-coupled receptor (GPCR) signaling activates YAP and TAZ.25 Thus, we determined the effect of GGylation on LPA-induced activation of YAP and TAZ using the expression of the target gene CYR61, the protein level of TAZ and the nuclear translocation of YAP as readouts (Figure 3). LPA-induced CYR61 expression was strongly inhibited by GGTI-298 and atorvastatin (Figure 3a). Consistently, LPA-induced enhancement of TAZ was eliminated by both GGTI-298 and atorvastatin, and LPA-induced nuclear translocation of YAP was diminished by GGTI-298 (Figures 3b and c). These data demonstrate that GGylation is essential for LPA-induced activation of YAP and TAZ in MDA-MB-231 cells.

Figure 3
figure3

Blockade of GGylation inhibits LPA-induced expression of TAZ and the YAP/TAZ target genes, and nuclear localization of YAP. (a) LPA-induced CYR61 expression is inhibited by the blockade of GGylation. HEK293A cells were starved and simultaneously treated with GGTI-298 or atorvastatin (Atv) for 24 h, and then stimulated for 1 h with LPA (1 μM, the same hereafter). (b) LPA-induced increase in TAZ amount is inhibited by blockade of GGylation. MDA-MB-231 cells were starved and simultaneously treated with DMSO, GGTI-298 or Atv for 24 h, and then stimulated for 1 h with LPA. TAZ was detected by direct immunoblotting and immunoprecipitation. (c) Blockade of GGylation by GGTI-298 abolished LPA-induced nuclear localization of YAP. MDA-MB-231 cells were starved and simultaneously treated with DMSO or GGTI-298 overnight, and then stimulated by LPA for 1 h. Scale bar, 20 μm. Graph displays the percentage of cells with nuclear YAP. Data are representative of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 (t-test); mean±s.e.m.

GGylation-dependent breast cancer cell proliferation and migration are mediated by the Hippo-YAP/TAZ pathway

To determine whether GGylation-dependent cell proliferation and migration is mediated by the Hippo pathway, we first examined whether YAP and TAZ participate in cell proliferation and migration in MDA-MB-231 cells. YAP or TAZ was knocked down using the short hairpin RNAs (shRNAs) (Figures 4a and d). Interestingly, knockdown of TAZ significantly inhibited cell migration, but had a minor inhibitory effect on proliferation in MDA-MB-231 cells (Figures 4b and c). Conversely, knockdown of YAP had a minor inhibitory effect on migration, but strongly suppressed proliferation of MDA-MB-231 cells (Figures 4e and f). As blockade of GGylation by GGTI-298 or atorvastatin inhibited both cell proliferation and migration (Figure 1), downregulated TAZ amount and diminished YAP translocation to the nuclei (Figure 2), these data suggest that YAP might mediate the effect of GGylation on the proliferation and TAZ on the migration of MDA-MB-231 cells.

Figure 4
figure4

Knockdown of YAP/TAZ inhibit MDA-MB-231 cells proliferation and migration. (a and d) TAZ or YAP expression was detected by western blotting. Stable shRNA cell lines were treated by doxycycline (1 μg/ml) for 2 days and YAP/TAZ were analyzed by western blotting. (b and e) Effect of TAZ or YAP knockdown on the proliferation of MDA-MB-231. Stable shRNA cell lines were treated by doxycycline (1 μg/ml) for 2 days. Then, cells were trypsinized and seeded in a 96-well plate for growing 48 h in the medium containing doxycycline. Cell proliferation was determined by MTT assay. (c and f) Effect of TAZ or YAP knockdown on the migration of MDA-MB-231. Stable shRNA cell lines were treated by doxycycline (1 μg/ml) for 2 days. Then, cells were trypsinized and seeded in the upper chamber containing doxycycline. After 24 h, transwell cell migration assay was performed. Data are representative of at least three independent experiments. **P<0.01 and ***P<0.001 (t-test); mean±s.e.m.

To verify the dependency of GGylation-regulated proliferation and migration on the Hippo-YAP/TAZ signaling, we searched for a breast cancer cell line with inactive YAP/TAZ signaling to examine whether the GGTI and atorvastatin inhibit proliferation and migration in this cell line. We found that MCF-7, an ER-positive breast cancer cell line, has extremely low mRNA expression of CTGF and CYR61 compared with MDA-MB-231 cells (Figure 5a). Consistently, expression of YAP/TAZ is also much less than that in MDA-MB-231 cells (Figure 5b). Furthermore, in contrast to MDA-MB-231 cells, serum stimulation did not induce translocation of YAP to the nuclei and enhance the expression level of TAZ in MCF-7 cells (Figure 5c). These data indicate that YAP/TAZ signaling in MCF-7 cells is inactive and does not respond to extracellular stimuli.

Figure 5
figure5

Blockade of GGylation has an insignificant effect in MCF-7 cells that are defective in the Hippo-YAP/TAZ signaling. (a) MCF-7 cells have extremely low expression of CTGF and CYR61. The mRNA level of CTGF and CYR61 was measured in both cell lines by qRT–PCR. (b) MCF-7 cells have much lower expression of TAZ and YAP. TAZ was detected by immunoprecipitation (IP) and YAP by direct immunoblotting. (c) YAP nuclear localization and TAZ amount is induced by the serum (10%) in MDA-MB-231 cells, but not in MCF-7. MDA-MB-231 and MCF-7 cells were starved overnight, followed by serum stimulation for 1 h. Scale bar, 20 μm. (d) Blockade of GGylation by GGTI-298 had an insignificant effect on CTGF and CYR61 expression in MCF-7 cells. (e) The effect of GGylation on the proliferation of MCF-7 cells was determined by the MTT assay. (f) The effect of GGylation on cell migration was determined by the wound healing assay. Data are representative of at least three independent experiments. ***P<0.001 (t-test); mean±s.e.m.

Next, we tested whether GGylation signals to the Hippo-YAP/TAZ pathway in MCF-7 cells. Analysis by qRT–PCR indicated that GGTI-298 treatment had a minor effect and atorvastatin had no effect on expression of CTGF and CYR61 (Figure 5d). We further examined the effect of GGylation on proliferation and migration of MCF-7 cells. As shown in Figure 5e, GGTI-298 treatment resulted in a slight inhibition of proliferation and atorvastatin had no effect on proliferation in MCF-7 cells. The wound healing assay demonstrated that neither GGTI-298 nor atorvastatin treatment affected migration of MCF-7 cells (Figure 5f). These results strongly suggest the GGylation-regulated breast cancer cell proliferation and migration are dependent on the Hippo-YAP/TAZ signaling.

To establish the correlation of GGylation signaling with the Hippo-YAP/TAZ signaling in breast cancer, we collected nine breast cancer cell lines to examine the effect of GGTI-298 on both proliferation and expression of CYR61 in parallel (Figure 6). As shown in Figures 6a and b and Supplementary Figure S2, the effect of GGTI-298 on inhibition of CYR61 expression is very well correlated with the effect on inhibition of the cell proliferation in these nine breast cancer cell lines. The calculated coefficiency index is as high as 0.8915 (Figure 6c), suggesting that the GGylation-mediated cell proliferation in breast cancer cells is largely dependent on the Hippo-YAP/TAZ signaling. Interestingly, treatment of T47D cells with GGTI-298 markedly increased the expression level of CYR61 (Figure 6a), but not proliferation (Figure 6b). However, the molecular mechanism by which GGTI-298 induces the expression of CYR61 in T47D cells is unknown.

Figure 6
figure6

GGylation-dependent proliferation is correlated with CYR61 expression in breast cancer cell lines. (a) The effects of GGTI-298 on CYR61 expression in MDA-MB-468, HCC1143, MDA-MB-231, SKBR3, HCC1569, MCF-7, MBA-MD-453, HCC1500 and T47D cells. The cells were treated with DMSO or GGTI-298 for 24 h. The mRNA level of CYR61 was quantified by qRT–PCR and normalized to the same level in the controls for comparison. Values of the unnormalized level of CYR61 mRNA with or without GGTI-298 treatment in these breast cancer cells are shown in Supplementary Figure S2. (b) The effect of GGTI-298 on cell proliferation of MDA-MB-468, HCC1143, MDA-MB-231, SKBR3, HCC1569, MCF-7, MBA-MD-453, HCC1500 and T47D cells. Cells were treated with GGTI-298 for 48 h and cell proliferation was determined by the MTT assay. (c) The correlation between the inhibition effects of GGTI-298 on proliferation and CYR61 expression in nine breast cancer cell lines. The best-fit linear trendline and the correlation coefficient were calculated and displayed by Microsoft Excel. Data are representative of at least three independent experiments. NS, not significant (P>0.05); *P<0.05; **P<0.01 and ***P<0.001 (t-test); mean±s.e.m.

Gβγ and RhoA may be direct effectors of GGylation for signaling to the Hippo pathway

We next searched for the direct effectors of GGylation for mediating signaling to the Hippo-YAP/TAZ pathway. We have shown that GGylation mediates LPA-induced activation of YAP and TAZ (Figure 3). In the LPA-stimulated GPCR signaling, Gγ-subunit and RhoA are the known GGylated proteins involved in cell adhesion, migration and proliferation.1,26, 27, 28 We first examined whether Gγ-subunits mediate the GGylation-dependent activation of YAP and TAZ. Gallein is a known inhibitor that binds to Gβγ and inhibits Gβγ function, while the gallein analog fluorescein has no effect on Gβγ.29 As shown in Figure 7a, gallein treatment significantly reduced the expression of CTGF and CYR61 in MDA-MB-231 cells, suggesting that Gβγ mediates the activation of YAP and TAZ. Consistently, gallein also inhibited nuclear localization of YAP and the LPA-induced enhancement of TAZ protein level, while fluorescein had no effect (Figures 7b and c). Furthermore, gallein inhibited cell proliferation and migration in MDA-MB-231 cells by about 30% and 50%, respectively (Figures 7d–f). In contrast, gallein treatment has no effect on YAP/TAZ signaling in MCF-7 cells (Supplementary Figure S3A), and did not inhibit cell proliferation and migration in MCF-7 cells (Supplementary Figures S3B and S3C). Similarity of the effects of gallein on the Hippo-YAP/TAZ signaling and cell proliferation and migration to that of GGTI-298 and atorvastatin suggests that Gβγ might participate in GGylation signaling and mediate the GGylation-dependent activation of YAP/TAZ.

Figure 7
figure7

The Gβγ inhibitor gallein inhibits the YAP/TAZ signaling. (a) Gallein treatment inhibited CTGF and CYR61 genes expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with DMSO or gallein (100 μM, the same as below) for 24 h. mRNA levels of CYR61 and CTGF were quantified by qRT–PCR. (b) Gallein inhibited YAP nuclear localization. MDA-MB-231 cells were treated with DMSO, fluorescein or gallein for 24 h. Scale bar, 20 μm. (c) Gallein abolished the effect of LPA-induced increase in TAZ amount. MDA-MB-231 cells were starved and treated with DMSO, fluorescein or gallein overnight, followed by stimulation with LPA for 1 h. (d) Gallein inhibited MDA-MB-231 cells' proliferation. (e and f) Gallein inhibited MDA-MB-231 cells' migration. Data are representative of at least three independent experiments. *P<0.05 and **P<0.01 (t-test); mean±s.e.m.

We further investigated whether a specific type of Gβγ-subunit mediates the GGylation-dependent activation of YAP and TAZ. Our GeneChip microarray data have shown that γ5 is the major form of the γ-subunits expressed in MDA-MB-231 cells. Thus, we tested if γ5 mediated the GGylation-dependent activation of YAP and TAZ using the HA-tagged non-prenylated mutant γ5-SSFL. As shown in Supplementary Figure S4A, inhibition of GGylation in MDA-MB-231 cells by GGTI-298 treatment eliminated the localization of γ5-CSFL (wild-type) on plasma membrane. Consistently, the non-prenylated mutant γ5-SSFL had no plasma membrane localization (Supplementary Figure S4B). These data indicate that the GGylation is required for γ5 to localize on the plasma membrane. However, no significant effect of overexpression of the wild-type or the mutant γ5-SSFL on the Hippo pathway was observed in MDA-MB-231 cells (Supplementary Figure S4C). Likewise, γ5 knockdown also had no detectable effect on the YAP/TAZ pathway in MDA-MB-231 cells (Supplementary Figure S4D), and as expected, no inhibitory effect on the cell proliferation and migration (data not shown). There are five types of Gβ-subunits and 12 types of Gγ-subunits in mammalian cells.30 Eight of 12 Gγ-subunits (γ2, γ3, γ4, γ5, γ7, γ10, γ12 and γ13) are putatively GGylated.1 Thus, it is possible that signaling from multiple Gβγ-subunits contributes to the activation effect. We may need to use RNA interference or shRNA to screen all the GGylated γ-subunits for the identification of the effector γ-subunits in future studies.

RhoA has been reported to mediate LPA-activated YAP/TAZ signaling.31 Thus, we first examined whether RhoA mediates GGylation-dependent activation of YAP-TAZ. As shown in Figure 8a, C3, a specific inhibitor of Rho GTPase, markedly inhibited CYR61 expression in MDA-MB-231 cells. Consistently, C3 partially inhibited migration and proliferation of MDA-MB-231 cells (Figures 8b and c). Next, we examined whether RhoA is the effector of GGylation for signaling to the Hippo-YAP/TAZ pathway in MDA-MB-231 cells. As cellular function of RhoA is dependent on prenylation but independent of the prenylation type (farnesylation or GGylation),32 we expected that overexpression of Fylated RhoA (RhoA-CVLS) would be capable of reversing the effect of GGTI-298 treatment on the Hippo-YAP/TAZ signaling. As expected, treatment with the GGTI-298 caused a redistribution of wild-type (RhoA-CLVL) from membrane to a diffuse cytoplasmic location, whereas the distribution of Fylated RhoA (RhoA-CVLS) was still on membrane (Figure 8d). Consistently, overexpression of the Fylated RhoA partially rescued the inhibitory effect of GGTI-298 on CYR61 expression in MDA-MB-231 (Figure 8e). Likewise, the Fylated RhoA bypassed GGylation and also partially rescued inhibitory effect of GGTI-298 on cell proliferation (Figure 8f). These results suggest that RhoA is one of the effectors mediating GGylation-dependent signaling of the Hippo pathway, which is consistent with the observation that Gβγ-subunits also participate in this pathway.

Figure 8
figure8

Inhibition of RhoA mimics the effect of GGylation inhibition on the YAP/TAZ signaling. (a) Rho GTPase inhibitor C3 markedly inhibited the CYR61 expression in MDA-MB-231 cells. The cells were separately treated with solvent and C3 (0.1 μM) for 24 h. (b) The RhoA inhibitor C3 inhibited MDA-MB-231 cells' proliferation. The cells were seeded and cultured for 24 h, and then separately treated with solvent and C3 for 48 h. Cell proliferation was determined by the MTT assay. (c) The effect of C3 on cell migration was determined by the wound healing assay. (d) Subcellular localization of transiently transfected wild-type RhoA (RhoA-CLVL) and Fylated RhoA (RhoA-CVLS) in HEK293A cells after treatment with GGTI-298 for 24 h. Scale bar, 20 μm. (e) The inhibitory effect on CYR61 expression by GGTI-298 was partially rescued by the expression of Fylated RhoA (RhoA-CVLS). MDA-MB-231 stable cells expressing GFP, wild-type RhoA or RhoA-CVLS were treated with DMSO and GGTI-298 for 24 h. mRNA level of CYR61 were quantified by qRT–PCR. (f) The inhibitory effect on cell proliferation by GGTI-298 was partially rescued by expression of Fylated RhoA (RhoA-CVLS). Cell proliferation was determined by the MTT assay. Data are representative of at least three independent experiments. *P<0.05 and **P<0.01 (t-test); mean±s.e.m.

Discussion

We have demonstrated that blockade of GGylation inhibits breast cancer cells' proliferation and migration through impairing the highly conserved the Hippo-YAP/TAZ signaling pathway, which has emerged as one prominent pathway in many types of cancers. Based on the results, we propose a GGylation-dependent signaling pathway that regulates cancer cell proliferation and migration by the inactivation of the Hippo signaling and the activation of Yap/Taz as shown in Figure 9. The GGylation of Gγ and RhoA controls GPCR-initiated signaling (such as LPA receptor-initiated signaling) to the Hippo pathway, which is mediated by both Gα and Gβγ. This GGylation signaling results in the activation of the Hippo pathway effectors YAP and TAZ and expression of the target genes involved in cell proliferation and migration. YAP activation may contribute mainly to the proliferation, while TAZ activation may contribute mainly to migration. Currently, how Gβγ or RhoA signals to the Hippo pathway is still unclear.

Figure 9
figure9

A model depicting how GGylation mediates the Hippo-YAP/TAZ signaling that regulates breast cancer cells' proliferation and migration. Gβγ and RhoA are the direct GGylated effectors signaling to the Hippo pathway. The major effectors of the Hippo signaling pathway, YAP and TAZ, regulate the expression of the target genes, such as CYR61 and CTGF, to produce effects on cell proliferation and migration. YAP is mainly attributed to the proliferation, while TAZ mainly attributed to migration.

Hippo protein (Mst1/2 in mammalian) was initially identified as a negative regulator in cell proliferation and organ size control in Drosophila. Inactive mutation of the Hippo protein induces uncontrolled organ overgrowth in Drosophila.33,34 The Hippo pathway regulates mammalian cell proliferation and migration through its effectors YAP and TAZ, two transcriptional coactivators.35,36 The target genes of YAP/TAZ involved in proliferation and migration include BIRC5, CYR61 and CTGF.37, 38, 39 Overexpression of YAP/TAZ induces transformation in mammalian cells and promotes tumorigenesis in transgenic mice models.40,41 Numerous studies have demonstrated overexpression of YAP/TAZ, as well as altered activity of the Hippo pathway, in a wide range of human cancers.42, 43, 44, 45 Further, several studies have demonstrated that most highly invasive breast cancer cell lines express TAZ at levels that are approximately four times of those expressed by the majority of weakly invasive breast cancer cells, and YAP activity is strongly correlated with breast cancer cells' metastatic potential.36,46 Our studies have connected the GGylation signaling to the Hippo-YAP/TAZ signaling pathway, suggesting that the GGylation-dependent cell survival and migration might be a marker of breast cancer progression through the activation of YAP/TAZ signaling.

Recently, two other studies have demonstrated that mevalonate pathway controls YAP/TAZ signaling via biosynthesis of GGPP,47,48 consolidating our findings about the role of GGylation in the activation of the YAP/TAZ signaling. However, there is a discrepancy in our results about the effects of statins and GGylation on the Hippo pathway, particularly the phosphorylation of Lats1/2. Our data have shown that inhibition of mevalonate pathway by atorvastatin or GGylation by GGTI-298 in MDA-MB-231 cells increased phosphorylation of Mst1/2 and Lats1 (Figure 2c), which are the upstream kinases of YAP/TAZ in the established Hippo signaling,35,36 suggesting that GGylation regulates the Hippo signaling. The other two studies found that the mevalonate pathway-mediated YAP/TAZ signaling was independent of Lats1/2 using Lats1/2 siRNA knockdown approaches.47,48 This discrepancy may be produced by differences in the experimental approaches. Further investigation should be carried out to verify the role of mevalonate pathway or GGylation in the regulation of the Hippo pathway in breast cancer cells.

Mammary tumors contain multiple sub-populations of tumor cells with distinct genotypic and phenotypic characteristics. During tumor progression, phenotypic characteristics of different sub-populations can be transformed by switching intracellular signaling pathways on or off, or by switching from one signaling pathway to another. The genotypic characteristics associated with the phenotypic characteristics determine cancer progression, such as metastatic potential and therapeutic response.49, 50, 51 Our studies suggest that GGylation signaling is an important factor in determination of the breast cancer phenotypic characteristics associated with the cancer progression. As shown in Figure 6, highly invasive human breast cancer cell lines MDA-MB-231, MDA-MB-468 and HCC1143 were sensitive to GGTI treatment, whereas luminal breast cancer cell lines with low invasive potential, such MCF-7 and T47D, were resistant to GGTI treatment. This implies that GGylation signaling may evolve to become a major survival pathway in highly invasive breast cancer cells, but not in weakly invasive or noninvasive breast cancer cells such as MCF-7 cells.

Statins, inhibitors of the rate-limiting enzyme 3-hydroxyl-3-methylglutaryl-CoA reductase in the mevalonate pathway, have been used in multiple preclinical models of breast cancer.52,53 Interestingly, one epidemiologic study reported that statins have a specific protective effect in breast cancer. The incidence of hormone receptor-negative (estrogen receptor-negative/progesterone receptor-negative) tumors in patients taking statins significantly decreased, but no such effect was observed for hormone receptor-positive tumors.54 In either breast cancer cell lines or mouse models of breast cancer, statins have a more marked role in the inhibition of hormone receptor-negative cancers.21,22 The majority of estrogen receptor-negative/progesterone receptor-negative breast tumors are highly invasive. Because the Hippo tumor suppressor signaling pathway is usually ‘switched off’ in highly invasive breast cancer, statins may reactivate the Hippo pathway in these breast tumors through the inhibition of GGylation.

A growing body of evidence indicates that GPCRs are involved in breast tumor progression. It has been shown that inhibition of Gβγ signaling suppressed breast tumor growth and metastasis both in vitro and in vivo.55 Many signaling molecules in cell motility and survival, including phosphatidylinositol 3-kinase, the small GTPases Rac, Cdc42 and PLCβ, are regulated by Gβγ.56 A recent study shows that blocking Gβγ signaling prevents SDF1α-induced breast tumor cell migration and invasion in vitro.57 Consistent with this, our studies have shown a significant effect of Gβγ inhibitor treatment on inhibition of MDA-MB-231 migration and suggest that the Hippo-YAP pathway is the downstream signaling pathway of Gβγ. Inhibition of Gβγ signaling inactivates the Hippo pathway effectors YAP and TAZ and reduces breast cancer cell proliferation and migration. This Gβγ signaling is a new regulatory pathway for breast cancer cells migration. The Hippo-YAP pathway has been identified as a downstream branch of GPCR signaling.25 They proposed that a wide range of G proteins can potentially influence the Hippo-Yap pathway. Our studies have shown that not only Gα but also Gβγ may transduce the GPCR-initiated Hippo-YAP signaling, dependent on GGylation signaling, adding a new scenario to the Hippo-YAP/TAZ signaling.

Breast cancer is the most commonly diagnosed cancer and the major cause of cancer-related mortality in women worldwide. Systematic characterization of breast cancer genomes has identified that different subtypes of breast cancer have somatic mutations in different signaling pathways. For example, the luminal breast cancer MCF-7 cells have a somatic mutation of PIK3CA. The cell survival is mainly dependent on the phosphatidylinositol 3-kinase activity,58 which makes the cells generally more sensitive to inhibitors of phosphatidylinositol 3-kinase than other signaling inhibitors (such as GGTIs). Our results and previous studies have demonstrated that the effectors YAP and TAZ are more active in highly invasive breast cancer cells than weakly invasive breast cancer cells and have a critical role in proliferation and migration. This suggests an addiction to the Hippo-YAP pathway in basal-like breast cancer cell lines, such as MDA-MB-231, MDA-MB-468 and HCC1143.46 Here we identify the Hippo-YAP pathway as a downstream mediator of GGylation-dependent cell proliferation and migration in highly invasive breast cancer cells, but not in weakly invasive breast cancer cells such as MCF-7. These highly invasive breast cancer cell lines are much more sensitive to GGTI-298. This indicates that a breast tumor that is addicted to the Hippo-YAP signaling could be highly aggressive and sensitive to statins and GGTIs. Thus, in the breast cancer clinic, targeting GGylation might be an effective therapeutic approach for patients with the YAP/TAZ-activating tumors marked by the high expression of CYR61 or CTGF.

Materials and methods

Materials

GGTI-298, GGOH and FOH were purchased from Sigma (St Louis, MO, USA). Atorvastatin was from LC Laboratories (Woburn, MA, USA). Doxycycline was from MP Biomedicals (Santa Ana, CA, USA). Fluorescein and gallein were from TCI America (Portland, OR, USA). Rho inhibitor C3 was from Cytoskeleton Inc (Denver, CO, USA). Anti-Myc antibody was from Covance (Denver, PA, USA). Anti-TAZ mouse antibody was from BD Biosciences (San Jose, CA, USA). Anti-phospho-Mst1/2 (T183/T180), anti-Mst1, anti-Mst2, anti-Lats1, anti-phospho-Lats1 (S909) and anti-phospho-YAP (S127) were from Cell Signaling Technology Inc (Danver, MA, USA). Polyclonal anti-human YAP antibody was generated in rabbits as described previously.59 The shRNA-expressing lentiviral vector pLKO-Tet-On was purchased from Addgene (Cambridge, MA, USA). The RhoA plasmids pCMVintronA-RhoA and pCMVintronA-RhoA-CVLS (farnesylated mutant of RhoA) were kindly provided by Dr Pradines at the Département Innovation Thérapeutique et Oncologie Moléculaire, Centre de Physiopathologie, Institut Claudius Regaud (Toulouse, France). The retroviral plasmid pBabe-TRPV4 was a kind gift from Dr Steyger of Oregon Hearing Research Center (Portland, OR, USA).

Cell culture and establishment of stable cell lines

Breast cancer cell lines MDA-MB-468, HCC1143, MDA-MB-231, SKBR3, HCC1569, MCF-7, MBA-MD-453, HCC1500 and T47D were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). HEK293A was provided by Dr Kunliang Guan at University of California at San Diego (San Diego, CA, USA). The human breast carcinoma cell line MDA-MB-231 was transduced with lentiviral or retroviral vector-loaded shRNAs and selected with puromycin (2.5–5 μg/ml ) to establish stable knockdown cell lines.

Lentiviral and retroviral constructs and virus packaging

The synthesized oligos for shRNAs were annealed and inserted into the AgeI/EcoRI-digested pLKO-Tet-On vector. A scramble shRNA oligo, which does not match any known human cDNA, was used as the control shRNA. The targeting sequences used were as follows: TAZ shRNA, 5′-IndexTermCCAGGAACAAACGTTGACTTA-3′ (shTAZ-1) and 5′-IndexTermGCCCTTTCTAACCTGGCTGTA-3′ (shTAZ-2); YAP shRNA, 5′-IndexTermCCCAGTTAAATGTTCACCAAT-3′ (shYAP-1) and 5′-IndexTermGCCACCAAGCTAGATAAAGAA-3′ (shYAP-2); Gγ5 shRNA, 5′-IndexTermCTGGATAACATTTGCTCCATT-3′ (shG5-1) and 5′-IndexTermCGAGCAACTTAAGAACCAGAT-3′ (shG5-2); and control shRNA, 5′-IndexTermCCTAAGGTTAAGTCGCCCTCG-3′ (control shRNA).

Human Gγ5 cDNA was amplified by PCR from the plasmid pcDNA3-HA-γ5 and ligated into BamHI and SalI sites of the retroviral vector pBabe. The non-prenylated γ5 mutant HA-γ5-SSFL was generated by mutating the C in the CAAX motif into S using the Quikchange Site-Directed Mutagenesis Kit (Stratagene). pBabe-puro-RhoA and pBabe-puro-RhoA-CVLS (farnesylated mutant) were constructed by PCR using pCMVintronA-RhoA or pCMVintronA-RhoA-CVLS as the template. The amplified RhoA fragments digested by BamHI and SalI were ligated into the retroviral vector.

Lentiviral or retroviral particles were packaged by transfecting HEK293T cells with the pLKO-Tet-On vector or pBabe vector together with packaging plasmids psPAX2 (encoding gag, pol and rev) or pUMVC and pMD2.G. The supernatant of the culture medium containing the viral particles was collected on days 2 and 3 after transfection. Viral particles were concentrated by ultracentrifugation (60 000 g for 1 h).

RNA extraction, reverse transcription and real-time PCR

After treatment, cells were washed with cold phosphate-buffered saline and subjected to RNA extraction using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA samples (1 μg) were reverse transcribed to complementary DNA using QuantiTect Reverse Transcription Kit (Qiagen). Complementary DNA was then diluted and used for quantification (with GAPDH gene as a control) by real-time PCR, which was performed using SYBR Select Master Mix (Applied Biosystems) and the 7500 fast Real-time PCR system (Applied Biosystems, Grand Island, NY, USA). Primers used in this study were as follows: GAPDH, 5′-IndexTermGTCAAGGCTGAGAACGGGAA/AAATGAGCCCCAGCCTTCTC-3′; CTGF, 5′-IndexTermATGGTGCTCCCTGCATCTTC/CTGGTACTTGCAGCTGCTCT-3′; CYR61, 5′-IndexTermCCAGTGTACAGCAGCCTGAA/CGCATCTTCACAGTCCTGGT-3′; Gγ5, 5′-IndexTermATGTCTGGCTCCTCCAGCGT/CTACAAAAAGGAACAGACTTTCT-3′.

Determination of cell proliferation

Cell proliferation was detected by two methods: the cell counting assay and the MTT assay.

The cell counting assay

Breast cancer cells were seeded and cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS at 37 °C in 5% CO2 for 24 h. The cells were visualized under a phase-contrast microscope and counted with a cell counter (Cellometer Auto T4; Nexcelom, Lawrence, MA, USA) in triplicate.

The MTT assay

Breast cancer cells were seeded in a 96-well plate at a density of 10 000 cells per well and incubated for 24 h. The cells were treated with the indicated concentrations of inhibitors and incubated for 48 h. Then, the medium was aspirated and replaced with complete medium containing 5 mg/ml MTT and incubated for 4 h at 37 °C in 5% CO2 humidified incubator. The medium was aspirated and MTT solvent (4 mM HCl, 0.1% Nonidet P-40 all in isopropanol) was added. The plate was covered with tinfoil and the cells were agitated on orbital shaker for 15 min. Absorbance at 590 nm was read with a reference filter of 620 nm. For inducible shRNA stable cell lines, doxycycline (1 μg/ml) was added to the medium to induce shRNA expression for 48 h. Then, the cells were trypsinized and seeded in a 96-well plate and allowed to grow for 48 h in the medium containing doxycycline and assayed by MTT.

Cell migration assays

Cell migration was detected by two assays: the wound healing assay and the Transwell assay.

The wound healing assay

The wound healing assay of tumor cells was performed as described previously.60 Breast cancer cells grown to 100% confluent monolayer were pretreated with indicated concentration of inhibitors for 8 h. Wounds were created by scraping with a 10-μl pipette tip. Images of wounds were taken immediately and 14 h after wounding. Wound areas were measured by Photoshop software, and wound closure was calculated as the percentage of wound area change by quantification of the pixel numbers in the gap area using Adobe Photoshop CS5 (Adobe, San Jose, CA, USA).

The Transwell assay

The Transwell assay procedures were performed as described previously.61 Cell migration was assayed using Transwell chambers (6.5 or 12 mm; Corning, Corning, NY, USA) with 8 μm pore membranes. The lower chamber was filled with 600 μl of 20% FBS Dulbecco's modified Eagle's medium medium. After treatment by inhibitors (GGTI-298 and atorvastatin) for 24 h or doxycycline for 48 h, the cells (1 × 105) were suspended with 200 μl of the medium (Dulbecco's modified Eagle's medium with 1% FBS) and placed into the upper chamber with or without inhibitors. After 24 h, cells were fixed using 5% glutaraldehyde and stained using 0.5% crystal violet. Cells in the upper chamber were carefully removed, and cells that migrated through the membrane were assessed by photography. For quantification, crystal violet was extracted by methanol and the absorbance at 540 nm was measured.

Immunoprecipitation and immunoblotting

Immunoprecipitation and immunoblotting were performed as described previously.62 Briefly, the cell lysates were incubated with 20 μl of protein A beads (1:1 slurry) plus 1–4 μg of primary antibody for 4 h at 4 °C with rotation. The protein A beads were then washed with RIPA buffer three times, resuspended in 30 μl of sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer and used for gel electrophoresis. The proteins on the gel were transferred on to a polyvinylidene fluoride membrane and immunoblotted using a Western Lightning ECL Detection Kit (Perkin-Elmer, Waltham, MA, USA).

Immunofluorescence staining

The cells were cultured in glass coverslip-bottomed culture dishes (MatTek, Ashland, MA, USA) to 50–80% confluence. For LPA (1 μM) or FBS stimulation, the cells were serum starved for the indicated time of treatment. After the culture medium was aspirated, the cells were rinsed with phosphate-buffered saline two times, fixed with 3.7% paraformaldehyde at 25 °C for 30 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline at 25 °C for 20 min. After washing with PBST (Phosphate-buffered saline with 0.1% Tween-20), the cells were incubated with primary antibody at 37 °C for 1 h. Then, the cells were washed with PBST three times and incubated with secondary antibody that was conjugated with a fluorescent dye at 37 °C for 1 h. Finally, the cells were washed with PBST three times, and the immunofluorescence staining was visualized under Zeiss LSM710 confocal microscope (Carl Zeiss, Jena, Germany) or Nikon inverted fluorescent microscope (Nikon, Melville, NY, USA). YAP nuclear localization was determined based on predominant nuclear staining of YAP in 80–150 cells for each experiment (Figures 2e, 3c and 7b).

Microarray analysis

The mRNA expression profiles were determined by Affymetrix microarray analysis. MDA-MB-231 or MCF-7 cells were treated with DMSO, atorvastatin or atorvastatin plus GGOH for 40 h. The mRNA was isolated using Qiagen RNeasy mRNA Purification Kit (Hilden, Germany) and the cDNA was synthesized using Qiagen QuantiTect Reverse Transcription Kit (Hilden, Germany). The microarray assay was performed on GeneChip Human Gene 1.0 ST (Affymetrix, Santa Clara, CA, USA) by Weis Center Microarray facility.

Data analysis

(1) Statistical analysis: Data are presented as mean±s.e.m., and statistical comparisons between two groups were analyzed by two-tailed Student’s t-test (P<0.05 was considered as significant). (2) Calculation of the effect of treatments on cell proliferation: The inhibitory effect of treatments on the proliferation rate was calculated using the formula: Ep=((NtN0)/(NcN0)) × 100%, where Ep is the effect on cell proliferation, Nt is the cell number in the sample cultured after t days, Nc is the cell number in the control sample cultured after t days and N0 is the cell number seeded at start culture day. Proliferation inhibition rate of treatments: Ei=(1−Ep) × 100%. If the proliferation inhibition rate is negative (it means facilitating cell proliferation), it is regarded as 0. (3) The best-fit linear trendline and the correlation coefficient index among the data points of inhibition of proliferation versus inhibition of CYR61 expression by GGTI-298 in breast cancer cell lines, which are used for showing correlation between GGylation and the Hippo-YAP/TAZ signaling, were calculated and displayed by Microsoft Excel software (Redmond, WA, USA).

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Acknowledgements

We thank Dr Pradines of the Département Innovation Thérapeutique et Oncologie Moléculaire, Centre de Physiopathologie, Institut Claudius Regaud for sending us wild-type and the farnesylated RhoA plasmids, Dr Steyger of Oregon Hearing Research Center for the retroviral plasmid pBabe-TRPV4 and Dr Kunliang Guan of University of California at San Diego for HEK293A cell line. This research was supported by Geisinger Research Large Grant (SRC-081, to WY) and NIH R01GM050369 (to CHB).

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Correspondence to W Yang.

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Mi, W., Lin, Q., Childress, C. et al. Geranylgeranylation signals to the Hippo pathway for breast cancer cell proliferation and migration. Oncogene 34, 3095–3106 (2015). https://doi.org/10.1038/onc.2014.251

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