Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

OBJECTIVE: To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. METHODS: Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). RESULTS: PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. CONCLUSION: PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death.

The orphan receptor TR3 participates in angiotensin II-induced cardiac hypertrophy by controlling mTOR signalling.

Angiotensin II (AngII) induces cardiac hypertrophy and increases the expression of TR3. To determine whether TR3 is involved in the regulation of the pathological cardiac hypertrophy induced by AngII, we established mouse and rat hypertrophy models using chronic AngII administration. Our results reveal that a deficiency of TR3 in mice or the knockdown of TR3 in the left ventricle of rats attenuated AngII-induced cardiac hypertrophy compared with the respective controls. A mechanistic analysis demonstrates that the TR3-mediated activation of mTORC1 is associated with AngII-induced cardiac hypertrophy. TR3 was shown to form a trimer with the TSC1/TSC2 complex that specifically promoted TSC2 degradation via a proteasome/ubiquitination pathway. As a result, mTORC1, but not mTORC2, was activated; this was accompanied by increased protein synthesis, enhanced production of reactive oxygen species and enlarged cell size, thereby resulting in cardiac hypertrophy. This study demonstrates that TR3 positively regulates cardiac hypertrophy by influencing the effect of AngII on the mTOR pathway. The elimination or reduction of TR3 may reduce cardiac hypertrophy; therefore, TR3 is a potential target for clinical therapy.

Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation.

In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

Akt phosphorylates the TR3 orphan receptor and blocks its targeting to the mitochondria.

Acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) phosphorylates and regulates the function of many cellular proteins involved in processes such as metabolism, apoptosis and proliferation. However, the precise mechanisms by which Akt promotes cell survival and inhibits apoptosis have been characterized in part only. TR3, an orphan receptor, functions as a transcription factor that can both positively or negatively regulate gene expression. We have reported previously that the translocation of TR3 from the nucleus to the mitochondria can elicit a proapoptotic effect in gastric cancer cells. In our present study, we demonstrate that Akt phosphorylates cytoplasmic TR3 through its physical interaction with the N-terminus of TR3. When coexpressed with Akt, TR3 mitochondrial targeting was blocked and this protein adopted a diffuse expression pattern in the cytoplasm. Moreover, Akt displayed an ability to disrupt the interaction of TR3 with Bcl-2, which is thought to be a critical requirement for mitochondrial TR3 to elicit apoptosis. Consistently, insulin was also found to induce the phosphorylation of TR3 and abolish 12-O-tetradecanoylphorbol-13-acetate-induced mitochondrial localization, which was dependent upon the activation of the phophatidylinositol-3-OH-kinase-Akt signaling pathway. Taken together, our current data demonstrate a unique role for Akt in inhibiting TR3 functions that are not related to transcriptional activity but that correlate with the regulation of its mitochondrial association. This may represent a novel signal pathway by which Akt exerts its antiapoptotic effects in gastric cancer cells, i.e. by regulating the phosphorylation and redistribution of orphan receptors.

Orphan receptor TR3 attenuates the p300-induced acetylation of retinoid X receptor-alpha.

Acetylation modification regulates the functions of histone and nonhistone proteins, including transcriptional activity, protein interaction, and subcellular localization. Although many nuclear receptors have been shown to be modified by acetylation, whether retinoid X receptors (RXRs) are acetylated and how the acetylation is regulated remains unknown. Here, we provide the first evidence of RXRalpha acetylation by p300 on lysine 145. Acetylation of RXRalpha by p300 facilitated its DNA binding and subsequently increased its transcriptional activity. Furthermore, we discovered that TR3, an orphan receptor, exerted a negative regulation on p300-induced RXRalpha acetylation. TR3 significantly reduced the p300-induced RXRalpha acetylation and transcriptional activity, and such inhibition required the interaction of TR3 with RXRalpha. Binding of TR3 to RXRalpha resulted in the sequestration of RXRalpha from p300. 9-cis retinoic acid, a ligand for RXRalpha, enhanced the association of RXRalpha with TR3, rather than acetylation of RXRalpha by p300. Biological function analysis revealed that the mitogenic activity of RXRalpha stimulated by p300 was acetylation dependent and could be repressed by TR3. Upon the treatment of 9-cis retinoic acid, RXRalpha was translocated with TR3 from the nucleus to the mitochondria, and apoptosis was induced. Taken together, our data demonstrate the distinct regulatory mechanisms of p300 and TR3 on RXRalpha acetylation and reveal a previously unrecognized role for orphan receptor in the transcriptional control of retinoid receptors.

p53 mediates the negative regulation of MDM2 by orphan receptor TR3.

MDM2 is an oncoprotein whose transforming potential is activated by overexpression. The expression level of MDM2 is negatively regulated by orphan receptor TR3 that mainly acts as a transcriptional factor to regulate gene expression. However, the underlying mechanism is largely unclear. Here, we present the first evidence that inhibition of TR3 on MDM2 is mediated by p53. We found that TR3 directly interacts with p53 but not MDM2, and such interaction is critical for TR3 to inhibit MDM2 expression. TR3 downregulates p53 transcriptional activity by blocking its acetylation, leading to a decrease on the transcription level of MDM2. Furthermore, TR3 binding to p53 obstructs its ubiquitination and degradation induced by MDM2, resulting in the MDM2 ubiquitination and degradation. In addition, TR3 could enhance p53-mediated apoptosis induced by UV irradiation. Taken together, our findings demonstrate that p53 mediates the suppression of TR3 on MDM2 at both transcriptional and post-transcriptional level and suggest TR3 as a potential target to develop new anticancer agents that restrict MDM2-induced tumor progression.

RXRalpha acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.

Retinoid X receptor (RXR) plays a crucial role in the cross talk between retinoid receptors and other hormone receptors including the orphan receptor TR3, forming different heterodimers that transduce diverse steroid/thyroid hormone signaling. Here we show that RXRalpha exhibits nucleocytoplasmic shuttling in MGC80-3 gastric cancer cells and that RXRalpha shuttling is energy-dependent through a nuclear pore complex (NPC)-mediated pathway for its import and an intact DNA binding domain-mediated pathway for its export. In the presence of its ligand 9-cis retinoic acid, RXRalpha was almost exclusively located in the cytoplasm. More importantly, we also show that RXRalpha acts as a carrier to assist translocation of TR3, which plays an important role in apoptosis. Both RXRalpha and TR3 colocalized in the nucleus; however, upon stimulation by 9-cis retinoic acid they cotranslocated to the cytoplasm and then localized in the mitochondria. TR3 export depends on RXRalpha, as in living cells GFP-TR3 alone did not result in export from the nucleus even in the presence of 9-cis retinoic acid, whereas GFP-TR3 cotransfected with RXRalpha was exported out of the nucleus in response to 9-cis retinoic acid. Moreover, specific reduction of RXRalpha levels caused by anti-sense RXRalpha abolished TR3 nuclear export. In contrast, specific knockdown of TR3 by antisense-TR3 or TR3-siRNA did not affect RXRalpha shuttling. These results indicate that RXRalpha is responsible for TR3 nucleocytoplasmic translocation, which is facilitated by the RXRalpha ligand 9-cis retinoic acid. In addition, mitochondrial TR3, but not RXRalpha, was critical for apoptosis, as TR3 mutants that were distributed in the mitochondria induced apoptosis in the presence or absence of 9-cis retinoic acid. These data reveal a novel aspect of RXRalpha function, in which it acts as a carrier for nucleocytoplasmic translocation of orphan receptors.