Interplay of mevalonate and Hippo pathways regulates RHAMM transcription via YAP to modulate breast cancer cell motility

Abstract

Expression of receptor for hyaluronan-mediated motility (RHAMM), a breast cancer susceptibility gene, is tightly controlled in normal tissues but elevated in many tumors, contributing to tumorigenesis and metastases. However, how the expression of RHAMM is regulated remains elusive. Statins, inhibitors of mevalonate metabolic pathway widely used for hypercholesterolemia, have been found to also have antitumor effects, but little is known of the specific targets and mechanisms. Moreover, Hippo signaling pathway plays crucial roles in organ size control and cancer development, yet its downstream transcriptional targets remain obscure. Here we show that RHAMM expression is regulated by mevalonate and Hippo pathways converging onto Yes-associated protein (YAP)/TEAD, which binds RHAMM promoter at specific sites and controls its transcription and consequently breast cancer cell migration and invasion (BCCMI); and that simvastatin inhibits BCCMI via targeting YAP-mediated RHAMM transcription. Required for ERK phosphorylation and BCCMI, YAP-activated RHAMM transcription is dependent on mevalonate and sensitive to simvastatin, which modulate RHAMM transcription by modulating YAP phosphorylation and nuclear-cytoplasmic localization. Further, modulation by mevalonate/simvastatin of YAP-activated RHAMM transcription requires geranylgeranylation, Rho GTPase activation, and actin cytoskeleton rearrangement, but is largely independent of MST and LATS kinase activity. These findings from in vitro and in vivo investigations link mevalonate and Hippo pathways with RHAMM as a downstream effector, a YAP-transcription and simvastatin-inhibition target, and a cancer metastasis mediator; uncover a mechanism regulating RHAMM expression and cancer metastases; and reveal a mode whereby simvastatin exerts anticancer effects; providing potential targets for cancer therapeutic agents.

Keywords: actin assembly; crosstalk; metabolism; oncogene; tumor suppressor.

Fig. 1.

RHAMM is essential for BCCMI, and the mevalonate pathway regulates RHAMM expression, ERK activation, and BCCMI. (A) Western blot showing lentivirus-mediated shRNAs against RHAMM markedly knock down RHAMM expression in MDA-MB-231 cells. (B and C) Knockdown of RHAMM inhibited the migration (B) and invasion (C) of MDA-MB-231 cells. (D and E) The mevalonate pathway inhibitor simvastatin (Sim) significantly inhibited RHAMM mRNA (D) and protein (E) expression, whereas adding mevalonate (Meva) abolished the inhibitory effect. MDA-MB-231 cells were incubated with or without 250 μM mevalonate for 6 h before treatment with DMSO or 5 μM simvastatin for 24 h. (F and G) As in D and E but with 4T1 cells. (H) Simvastatin markedly inhibited RHAMM promoter activity, whereas adding mevalonate abolished the inhibition. MDA-MB-231 cells were transfected with RHAMM-Luc reporter plasmid for 6 h, and then treated with or without 250 μM mevalonate for 6 h before treatment with DMSO or 5 μM simvastatin for 24 h. Luciferase activity was measured and normalized to GAPDH. (I) Knockdown of RHAMM decreased ERK phosphorylation in MDA-MB-231 cells. (J) Simvastatin significantly inhibited ERK activity, whereas adding mevalonate abolished the inhibition. MDA-MB-231 cells were incubated with or without 250 μM mevalonate for 6 h before treatment with DMSO or 5 μM simvastatin for 24 h. Data are shown as the mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05).

Fig. 2.

YAP and TEAD bind to RHAMM promoter at specific sites and regulate RHAMM transcription. (A) The human RHAMM promoter region contains two putative TEAD-binding sites (TB; boxed). (B and C) YAP (B) and TEAD4 (C) bound to the RHAMM promoter by ChIP assay. ChIP from fragmented chromatins of MDA-MB-231 cells were incubated with IgG, anti-YAP or anti-TEAD4, or anti-RNA polymerase II (positive control; Con) antibody. (D) The putative TEAD-binding sites were important for YAP-mediated RHAMM promoter activity. (Upper) The two TEAD-binding sites were deleted individually or in combination. HEK 293T cells were cotransfected with control vector or V5-YAP in combination with WT or mutant RHAMM-Luc reporter plasmid. At 24 h after transfection, the luciferase activity was measured. (Lower) Western blot showing YAP protein level. (E) Knockdown of YAP or TEAD1/3/4 decreased RHAMM promoter activity. MDA-MB-231 cells were infected with the indicated shRNA lentiviruses, and luciferase activity (Upper) and protein levels (Lower) are shown. Data are shown as mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05 and **P < 0.01).

Fig. 3.

YAP and TEAD are required for RHAMM expression, ERK activity, and BCCMI, whereas ectopic expression of RHAMM bypasses the requirement. (A and B) Knockdown of YAP or TEAD1/3/4 decreased RHAMM transcription (A) and protein (B) levels. MDA-MB-231 cells were infected with the indicated shRNA lentiviruses, and RHAMM mRNA (A) and protein (B) levels were determined by real-time RT-PCR and Western blot, respectively. (B) (Right) Quantification of the protein bands. (C) Knockdown of YAP or TEAD1/3/4 reduced CTGF mRNA levels (experiments as in A). (D and E) Overexpression of YAP increased RHAMM mRNA (D) and protein (E) levels. HEK 293T cells were transiently transfected with the indicated YAP plasmids, and, 48 h later, total RNA was extracted to detect RHAMM mRNA by using real-time RT-PCR (D). (E) Cell lysates were immunoblotted with the indicated antibodies (Left), and the protein bands were quantified (Right). (F) Knockdown of YAP or TEAD1/3/4 decreased ERK phosphorylation levels. Experiments were as in B except Western blot was performed with the indicated antibodies. (G and H) Knockdown of YAP or TEAD1/3/4 decreased MDA-MB-231 cell migration (G, Left) and invasion (H, Left), whereas reexpression of RHAMM rescued the effects (G and H, Right). Data are shown as mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05 and ***P < 0.001).

Fig. 4.

YAP phosphorylation, cytoplasmic localization, and transactivating activity are modulated by mevalonate pathway and simvastatin (Sim). (A and B) Simvastatin significantly inhibited YAP activity (enhanced YAP phosphorylation), whereas adding mevalonate (Meva) abolished the effect in MDA-MB-231 (A) and 4T1 (B) cells. Cells were treated with or without 250 μM Meva for 6 h before incubation with DMSO or 5 μM simvastatin for 24 h and preparation of lysates, which were immunoblotted with p-YAP (Ser-127) and YAP antibodies, respectively (Left), and the protein bands quantified (Right). (A, Left) L, long exposure; S, short exposure. (C) Simvastatin significantly inhibited CTGF mRNA expression, whereas adding mevalonate abolished the inhibition in MDA-MB-231 cells. (D and E) Simvastatin induced YAP translocation from the nucleus to cytoplasm, whereas adding mevalonate prevented the YAP translocation in MDA-MB-231 (D) and 4T1 (E) cells. Immunofluorescent staining of YAP in the cells in the absence and presence of simvastatin and mevalonate. Cells were fixed and stained with YAP antibody (green) and DAPI (blue). (Right) Quantification of immunofluorescent images (ratio of nuclear YAP). (F) Western blot of nucleocytoplasmic fractionation indicated that simvastatin induced YAP translocation from the nucleus to cytoplasm, whereas adding mevalonate prevented the effect. CE, cytoplasmic extract; NE, nuclear extract. (G) Simvastatin prevented binding of YAP to RHAMM promoter. Chromatins were prepared from MDA-MB-231 cells incubated with or without 5 μM simvastatin. ChIP was carried out by using anti-YAP antibody, and the amplification was performed by using SYBR Green real-time PCR. Data are shown as the mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 5.

YAP and RHAMM are co-overexpressed in human breast invasive tumors, and simvastatin (Sim) modulates YAP phosphorylation, RHAMM expression, and ERK activity in the MDA-MB-231 human breast tumor xenografts in mice. (A) YAP and RHAMM are both overexpressed in human breast invasive ductal carcinoma. Tissue sections from breast invasive ductal carcinoma and cancer-adjacent normal breast tissue were immunostained with anti-YAP and anti-RHAMM antibody, and analyzed by light microscopy. (B and C) Simvastatin modulated YAP phosphorylation, RHAMM expression, and ERK activity in the MDA-MB-231 human breast tumor xenografts. (B) Simvastatin inhibited RHAMM mRNA expression in vivo. Total RNA was extracted from tumors from control (saline) or simvastatin-treated mice and then RHAMM mRNA levels were determined by real-time RT-PCR. (C) Simvastatin inhibited YAP activity, RHAMM protein expression, and ERK activity in vivo. Lysates of tumors from control (saline) or simvastatin-treated mice were immunoblotted with the indicated antibodies. Data are shown as the mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05).

Fig. 6.

Geranylgeranylation downstream of mevalonate pathway mediates regulation of YAP activity, RHAMM expression, and ERK activity. (A and B) GGTI-2133 inhibited RHAMM mRNA expression (A) and RHAMM protein expression and YAP and ERK activation (B). MDA-MB-231 cells were incubated with or without 1 μM GGTI-2133 for 24 h, and analyzed by real-time RT-PCR (A) and Western blot with the indicated antibodies (B). (C and D) As in A and B but with 4T1 cells. (E and F) GGTI-2133 induced translocation of YAP from the nucleus to the cytoplasm in MDA-MB-231 (E) and 4T cells (F). Cells were incubated with or without 1 μM GGTI-2133 for 24 h and fixed and stained with YAP antibody (green) and DAPI (blue) for immunofluorescent imaging. (G and H) GGPP rescued inhibition by simvastatin (Sim) of RHAMM mRNA expression (G) and RHAMM protein expression and YAP and ERK activity (H) in MDA-MB-231 cells. Cells were incubated with or without 25 μM GGPP for 2 h before treatment with DMSO or 5 μM simvastatin for 24 h, and analyzed by real-time RT-PCR (G) and Western blot with the indicated antibodies (H). (I and J) As in G and H but with 4T1 cells. (K and L) GGPP blocked translocation of YAP from the nucleus to the cytoplasm induced by simvastatin in MDA-MB-231 (K) and 4T1 cells (L). Cells were incubated with or without 25 μM GGPP for 2 h before treatment with DMSO or 5 μM simvastatin, and fixed and stained with YAP antibody (green) and DAPI (blue) for immunofluorescent imaging. Data are shown as the mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 7.

Mevalonate pathway regulates YAP activity, RHAMM expression, and ERK activity through Rho GTPase activity and actin cytoskeleton rearrangement largely independent of MST and LATS. (A and B) Constitutively active RhoA (Q63L) rescued RHAMM mRNA expression (A) and RHAMM protein expression, and YAP and ERK activity (B) decreased by simvastatin (Sim). MDA-MB-231 cells were infected with lentivirus-mediated constitutively active RhoA (Q63L)-EGFP, and, 24 h later, treated with simvastatin for another 24 h and analyzed by real-time RT-PCR (A) and Western blot with the indicated antibodies (B). (C–E) Disruption of actin cytoskeleton by various methods induced translocation of YAP from the nucleus to the cytoplasm (C), decrease in RHAMM mRNA level (D), and inhibition of RHAMM protein expression and YAP and ERK activation (E). MDA-MB-231 cells were treated with cytochalasin D or C3 transferase for 6 h or cultured on 1 kPa hydrogel for 24 h to disrupt actin cytoskeleton assembly, and then fixed and stained with phalloidin (green), YAP antibody (red) and DAPI (blue) for immunofluorescent imaging (C), or analyzed by real-time RT-PCR (D) or Western blot with the indicated antibodies (E). (F and G) MST phosphorylation (F) or LATS phosphorylation (G) was not modulated by mevalonate pathway/simvastatin, or actin cytoskeleton-disrupting cytochalasin D, C3 transferase, or culturing on 1 kPa hydrogel in MDA-MB-231 cells. (F) Cells were transfected with Flag-MST2 for 24 h and then treated with the indicated reagents, and cell lysates were immunoblotted with p-MST and Flag antibodies. The specificity of the p-MST antibody was confirmed by λ-phosphatase (PPase) treatment (lane 3). (G) Cell lysates were immunoblotted with p-LATS1 and LATS1 antibodies. The specificity of the p-LATS antibody was confirmed by λ-phosphatase treatment (lane 2). (H and I) Knockdown of LATS did not affect simvastatin-induced inhibition of RHAMM mRNA expression (H) or RHAMM protein expression and YAP and ERK activation (I). MDA-MB-231 cells were infected with the indicated shRNA lentiviruses, treated with or without simvastatin for 24 h, and then analyzed by real-time RT-PCR to determine RHAMM mRNA levels (H) or Western blot to detect the proteins (I). Data are shown as the mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 8.

Model of mevalonate and Hippo pathways regulating YAP-controlled RHAMM transcription and cancer cell motility. Mevalonate pathway regulates activity of YAP, the downstream effector of Hippo pathway, which activates transcription of RHAMM that is required for ERK activation and cancer cell migration and invasion. YAP/TEAD activates RHAMM transcription by binding to RHAMM promoter at two specific TEAD-binding sites. Mevalonate pathway promotes and simvastatin inhibits YAP activity, and consequently RHAMM transcription, ERK activation, and cancer metastasis, via modulating YAP phosphorylation and nuclear-cytoplasmic distribution. The regulation of YAP-mediated RHAMM transcription and cancer metastasis depends on geranylgeranylation, Rho GTPase activity, and actin cytoskeleton assembly, but not the canonical MST/LATS cascade.