XB-ART-59626
Cell Death Dis
2023 Jan 27;141:64. doi: 10.1038/s41419-022-05537-7.
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TPX2 enhances the transcription factor activation of PXR and enhances the resistance of hepatocellular carcinoma cells to antitumor drugs.
Wang H
,
Chu F
,
Zhang XF
,
Zhang P
,
Li LX
,
Zhuang YL
,
Niu XF
,
He X
,
Li ZJ
,
Bai Y
,
Mao D
,
Liu ZW
,
Zhang DL
,
Li BA
.
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The pregnane X receptor (PXR) is an important regulator of hepatocellular carcinoma cellular resistance to antitumor drugs. Activation of PXR was modulated by the co-regulators. The target protein for the Xenopus plus end-directed kinesin-like protein (Xklp2) known as TPX2 that was previously considered as a tubulin regulator, also functions as the regulator of some transcription factors and pro-oncogenes in human malignances. However, the actions of TPX2 on PXR and HCC cells are still unclear. In the present study, our results demonstrate that the high expression of endogenous mRNA level of TPX2 not only correlated with the poor prognosis of advanced HCC patients who received sorafenib treatment but also with expression of PXR's downstream genes, cyp3a4 and/or mdr-1. Results from luciferase and real-time polymerase chain reaction (qPCR) showed that TPX2 leads to enhancement of the transcription factor activation of PXR. Protein-protein interactions between PXR and TPX2 were identified using co-immunoprecipitation. Mechanically, overexpression of TPX2 led to enhancement of PXR recruitment to its downstream gene cyp3a4's promoter region (the PXRE region) or enhancer region (the XREM region). Treatment of HCC cells with paclitaxel, a microtubule promoter, led to enhancement of the effects of TPX2, whereas vincristine, a microtubule depolymerizing agent caused a decrease in TPX2-associated effects. TPX2 was found to cause acceleration of the metabolism or clearance of sorafenib, a typical tyrosine kinase inhibitor (TKI) in HCC cells and in turn led to the resistance to sorafenib by HCC cells. By establishing novel actions of TXP2 on PXR in HCC cells, the results indicate that TPX2 could be considered a promising therapeutic target to enhance HCC cells sensitivity to antitumor drugs.
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Genes referenced: abcb1 tpx2
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Fig. 1. TPX2, which is the target protein for the Xenopus plus end-directed kinesin-like protein (Xklp2), associates with the poor prognosis of advanced hepatocellular carcinoma (HCC) patients received sorafenib treatment.TPX2 was found to be related to the pregnane X receptor (PXR) pathway in HCC clinical specimens. (A and B) the expression of TPX2 in HCC clinical specimens (A and B) and the paired non-tumor tissues were examined using real-time polymerase chain reaction (qPCR), and the advanced HCC patients were divided into two groups: (1) TPX2 high group and (2) TPX2 low group according to the median values of TPX2. (C and D) Survival curves of advanced HCC patients were obtained and compared with the patient grouping and patient clinical follow-up information. The results are shown as overall survival (OS) (C) or time to progression (TTP) (D). (E–H) the expression level of TPX2, cyp3a4, mdr-1, PXR, or retinoid X receptor (RXR) in HCC clinical specimens was examined by qPCR. After that, a scatter plot was drawn based on the results of qPCR with the expression level of TPX2 as the abscissa (E–H), and the expression levels of cyp3a4 (E), mdr-1 (F), PXR (G), and RXR (H) as the vertical axis. The coordinates were plotted as scatter plots, which were then analyzed using linear regression. The endogenous mRNA expression of TPX2 (I) or PXR (J) in the selected hepatic cell lines (L-02, MHCC97-H, MHCC97-L, or HepG2 cells) was examined using qPCR. *P < 0.05. |
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Fig. 2. TPX2 enhances the transcription factor activation of ETS-1.(A) The downstream gene of PXR, cyp3a4, is upstream of the transcription initiation site, including the promoter region sequence (PXRE) and the enhancer region sequence (XREM). Two PXR binding sites (one DR3 motif and one ER6 motif) are found in XREM in addition to a PXR binding site (DR6 motif) in PXRE. (A) Schematic representation of the binding site of the cyp3a4 promoter to the enhancer region, PXR; schematic representation of the four luciferase reporters. The HCC cells, MHCC97-L (B–E), MHCC97-H (F–I), and HepG2 (J–M) were co-transfected with plasmids (control or TPX2 for MHCC97-L; control or siTPX2 for MHCC97-H; control, TPX2 or siTPX2 for HepG2 cells) (XREM-Luc [B, F and J]; PXRE-Luc [C, G and K]; DR3-Luc [D, H and L]; ER6-Luc [E, I and M]). Cells were treated with solvent control or rifampicin. The activation of XREM-Luc, PXRE-Luc, DR3-Luc, or ER6-Luc was examined by luciferase assays. (N-U) The HCC cells, MHCC97-L (N and O), MHCC97-H (P and Q) and HepG2 (R and S) were co-transfected with plasmids (control or TPX2 for MHCC97-L; control or siTPX2 for MHCC97-H; control, TPX2 or siTPX2 for HepG2 cells). Cells were treated with solvent control or rifampicin. The mRNA level of cyp3a4 (N, P and R) or mdr-1 (O, Q and S) was examined using qPCR. The effects of ketoconazole on TPX2 were shown (T and U). *P < 0.05. |
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Fig. 3. TPX2 interacts with PXR and affect the nuclear accumulation of PXR in HCC cells.(A and B) The interaction between TPX2 and PXR was identified by “IP: FLAG; IB: PXR” or “IP: FLAG; IB: TPX2”. (C and D) The interaction between FLAG-PXR mutation (including NTD [N-terminal domain, residues 1–40], DBD [DNA-binding domain, residues 41–110], HD [hinge domain, residues 1–140] and LBD [ligand-binding domain, residues 141–434]), and HA-TPX2; FLAG-TPX2 mutations (including 1–45aa, 46–140aa, 141–280aa, 281–320aa, and 321–747aa) and HA-PXR was identified by “IP: FLAG; IB: HA”. (E and F) The HCC cells, MHCC97-L (E), MHCC97-H (F), were co-transfected with plasmids (control or TPX2 for MHCC97-L; control or siTPX2 for MHCC97-H). Cells were treated with solvent control or rifampicin. The protein level of CYP3A4 or P-GP was examined using western blot. (G and H) The HCC cells, MHCC97-L (E), MHCC97-H (F), were co-transfected with plasmids (control or TPX2 for MHCC97-L; control or siTPX2 for MHCC97-H). Cells were treated with solvent control or rifampicin. The cells were separated into nuclear sub-fraction or cytoplasm sub-fraction. The protein level of PXR or TPX2 was examined using western blot. |
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Fig. 4. TPX2 enhances the recruitment of PXR to its downstream gene cyp3a4’s promoter or enhancer regions.(A) The downstream gene of PXR, cyp3a4, is upstream of the transcription initiation site, including the promoter region sequence (PXRE) and the enhancer region sequence (XREM). Two PXR binding sites (one DR3 motif and one ER6 motif) are located in XREM in addition to a PXR binding site (DR6 motif) in PXRE. In chromatin co-immunoprecipitation (ChIP), XREM and PXRE sequences were amplified respectively. The HCC cells, MHCC97-L (B and C), MHCC97-H (D and E) and HepG2 (F and G), were transfected with plasmids (control or TPX2 for MHCC97-L; control or siTPX2 for MHCC97-H; control, TPX2 or siTPX2 for HepG2 cells). Cells were treated with solvent control or rifampicin and analyzed using ChIP. P < 0.05. |
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Fig. 5. TPX2 promotes the metabolism or clearance of sorafenib in HCC cells or tumors.HCC cells MHCC97-L (A and B), MHCC97-H (C and D) or HepG2 (E and F) cells were cultured. The experiments were performed in cultured cells (A, C and E) or subcutaneous tumor tissues formed by HCC cells (B, D and F). The amount of sorafenib in cell or tumor samples was examined using liquid chromatography–mass spectrometry (LC–MS/MS). A retention curve of sorafenib was constructed according to the content of sorafenib measured in the samples at the 0 time point (baseline) and the content of sorafenib measured in the samples at each time point. *P < 0.05. |
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Fig. 6. TPX2 promotes the resistance of HCC cells to sorafenib in a subcutaneous tumor model.The HepG2 cells were transfected with control, TPX2, or siTPX2. Cells were then injected into the subcutaneous position of nude mice. The mice received 2.0 mg/kg (high dose), 1.0 mg/kg (medium dose) or 0.5 mg/kg (low dose) of sorafenib via oral administration. After treatment, the tumors were collected to measure the tumor volumes, tumor weights, and/or the mRNA level of genes (TPX2, PXR, cyp3a4 or mdr-1) by qPCR. The results were shown as images of subcutaneous tumors (A), histograms of tumor volumes/weights (B and C) or the heat-map of the mRNA level of genes (TPX2, PXR, cyp3a4, or mdr-1). *P < 0.05. |
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Fig. 7. TPX2 promotes the resistance of HCC cells to sorafenib in an intrahepatic subcutaneous tumor model.The HepG2 cells were transfected with control, TPX2 or siTPX2 and injected into the subcutaneous position of nude mice. The tumor tissues were prepared as the micro-blocks for constructing the intrahepatic HCC model. The mice received the 1.0 mg/kg concentration of sorafenib via oral administration. The results were shown as images of micro positron emission tomography (microPET) (A) or quantitative results. *P < 0.05. |
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Fig. 8. TPX2 promotes the recruitment of PXR to its downstream gene cyp3a4’s promoter or enhancer region.Rifampicin was used as an agonist of PXR, and the microtubule aggregation agent, paclitaxel, and the microtubule depolymerizing agent, vincristine, were used in combination with rifampicin. (A) The time points for this step are shown. Using a ChIP assay to detect the recruitment of PXR (B and C) or RXR in the enhancer (XREM) (B and D) and promoter (PXRE) (C and E) regions of cyp3a4. *P < 0.05. |
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