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. 2024 May 30;43(1):152.
doi: 10.1186/s13046-024-03070-3.

Unveiling the role of HP1α-HDAC1-STAT1 axis as a therapeutic target for HP1α-positive intrahepatic cholangiocarcinoma

Fei Xiong #  1   2 , Da Wang #  1 , Wei Xiong #  3 , Xin Wang  4 , Wen-Hua Huang  5 , Guan-Hua Wu  1 , Wen-Zheng Liu  1 , Qi Wang  1 , Jun-Sheng Chen  1 , Yi-Yang Kuai  1 , Bing Wang  6 , Yong-Jun Chen  7
Affiliations

Unveiling the role of HP1α-HDAC1-STAT1 axis as a therapeutic target for HP1α-positive intrahepatic cholangiocarcinoma

Fei Xiong et al. J Exp Clin Cancer Res. .

Erratum in

Abstract

Background: Intrahepatic cholangiocarcinoma (ICCA) is a heterogeneous group of malignant tumors characterized by high recurrence rate and poor prognosis. Heterochromatin Protein 1α (HP1α) is one of the most important nonhistone chromosomal proteins involved in transcriptional silencing via heterochromatin formation and structural maintenance. The effect of HP1α on the progression of ICCA remained unclear.

Methods: The effect on the proliferation of ICCA was detected by experiments in two cell lines and two ICCA mouse models. The interaction between HP1α and Histone Deacetylase 1 (HDAC1) was determined using Electrospray Ionization Mass Spectrometry (ESI-MS) and the binding mechanism was studied using immunoprecipitation assays (co-IP). The target gene was screened out by RNA sequencing (RNA-seq). The occupation of DNA binding proteins and histone modifications were predicted by bioinformatic methods and evaluated by Cleavage Under Targets and Tagmentation (CUT & Tag) and Chromatin immunoprecipitation (ChIP).

Results: HP1α was upregulated in intrahepatic cholangiocarcinoma (ICCA) tissues and regulated the proliferation of ICCA cells by inhibiting the interferon pathway in a Signal Transducer and Activator of Transcription 1 (STAT1)-dependent manner. Mechanistically, STAT1 is transcriptionally regulated by the HP1α-HDAC1 complex directly and epigenetically via promoter binding and changes in different histone modifications, as validated by high-throughput sequencing. Broad-spectrum HDAC inhibitor (HDACi) activates the interferon pathway and inhibits the proliferation of ICCA cells by downregulating HP1α and targeting the heterodimer. Broad-spectrum HDACi plus interferon preparation regimen was found to improve the antiproliferative effects and delay ICCA development in vivo and in vitro, which took advantage of basal activation as well as direct activation of the interferon pathway. HP1α participates in mediating the cellular resistance to both agents.

Conclusions: HP1α-HDAC1 complex influences interferon pathway activation by directly and epigenetically regulating STAT1 in transcriptional level. The broad-spectrum HDACi plus interferon preparation regimen inhibits ICCA development, providing feasible strategies for ICCA treatment. Targeting the HP1α-HDAC1-STAT1 axis is a possible strategy for treating ICCA, especially HP1α-positive cases.

Keywords: HDACi; HP1α; Histone modification; Interferon; STAT1.

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Conflict of interest statement

Not applicable.

Figures

Fig. 1
Fig. 1
HP1α downregulation impairs the proliferation of ICCA cells. A. The difference in HP1α expression among different subgroup in GSE32225. Subgroups were divided according to the pathological features. Inflammation group contained the samples characterized by the hyperactivation of inflammation pathways. Proliferation groups contained the samples featured by the overaction of oncogenic pathways and lack of inflammation response. B. IHC result of the tissue microarray of CCA using HP1α antibodies (28 CCA samples and 8 normal samples). C. IHC result of the ICCA tissue samples using HP1α antibodies (40 ICCA samples and 40 normal samples). D. The difference in HP1α expression between ICCA tissue and para-cancerous tissue evaluated by RT-qPCR (90 ICCA samples and 90 normal samples). E. Survival analysis of ICCA cohort. The median of HP1α expression level was selected as the cutoff value. F. The difference in HP1α expression among five ICCA cell lines. G. RT-qPCR analysis of the effect of HP1α knockdown. H. Western blot analysis of HP1α and proliferation and cell cycle-promoting factors. I. Evaluation of the impact on the distribution of cell cycle upon HP1α knockdown. J, K. Results of the proliferation assay upon HP1α knockdown. J. CCK-8 assay. K. Colony formation assays. L. Western blot analysis of HP1α expression, proliferation, and cell cycle-promoting factors upon HP1α overexpression. M. RT-qPCR analysis of the effect of HP1α overexpression. N. Evaluation of the impact on the distribution of cell cycle upon HP1α overexpression. O, P. Results of the proliferation assay following HP1α overexpression. O. CCK-8 assay. P. Colony formation assay. Blue bar, 20 μm. Red bar, 50 μm. Para-T: para-cancerous tissue. *P < 0.05; **P < 0.01
Fig. 2
Fig. 2
Downregulation of HP1α activates IFN signaling pathway. A. The correlation between the expression of MX1 and HP1α in the dataset of two distinct groups in GSE32225. B. RT-qPCR analysis of the expression of MX1, ISG15 and SAMHD1. C. Graphic illustration of the interventions mentioned in this section. PF-06700841 inhibited the IFN signaling by directly impairing the activation of JAK1 and TYK2. IRF9 could be knocked down by specific siRNAs. The activation level of IFN pathway could be indicated by the expression level of ISGs. D. RT-qPCR analysis of the expression of the upstream participants of IFN-I signaling E. The difference in STAT1 expression among five ICCA cell lines. F. Analysis of the correlation between the expression of HP1α and STAT1 in ICCA tissue (N = 90). G. Western blot analysis of the expression of the upstream participates of IFN-I signaling and the activation level of upstream kinases. H. Western blot analysis of the expression of STAT1 and p-STAT1 in cytoplasm and nucleus. I, J. Evaluation of the effect of inhibiting upstream kinases on the HP1α-STAT1 axis. I. RT-qPCR. J. Western blot. K, L. Effect of ISGF3 formation inhibition on the HP1α-STAT1 axis. K. RT-qPCR. L. Western blot. *P < 0.05; **P < 0.01
Fig. 3
Fig. 3
HP1α knockdown inhibits the proliferation of ICCA cells by upregulating STAT1 and IFN signaling induction. A, B. The results of GSEA analysis revealed the critical function of two genes based on the Reactome database with the dataset of the GSE32225 proliferation group. (A) STAT1. (B) HP1α. C. Evaluation of HP1α and STAT1 expression and IFN signaling activation by RT-qPCR upon STAT1 knockdown. D. Western blot analysis of HP1α and IFN signaling participants and proliferation-and cell cycle-promoting factors upon STAT1 knockdown. E, F. Results of the proliferation assay after STAT1 knockdown. E. Colony formation assay. White bar, 1.96 cm. F. CCK-8 assay. G. The distribution of the cell cycle of ICCA cells upon STAT1 knockdown. H. Presentation of the subcutaneous tumors of different groups. I. Evaluation of the Ki-67 positivity rate of subcutaneous tumors. Blue bar, 20 μm. Red bar, 50 μm. *P < 0.05; **P < 0.01
Fig. 4
Fig. 4
STAT1 is regulated by HDAC1 and histone acetylation. A. Summary of the histone marks located in the promoter region of STAT1 gene. B. Summary of the interaction between HDACs and HP1α. The combined score calculated by STRING was shown. HDACs were divided into three groups according to whether the interaction was previous found in human tissues. C. The heatmap of the RNA-seq data of TSA-treated ICCA cells. The gradual change from red to blue represents changes in gene expression from high to low. D. Western blot analysis of the total histone acetylation induced by TSA treatment. E. Western blot analysis of the total level of selected histone marks upon HP1α knockdown. F, G. Results of the proliferation assay upon TSA treatment and HP1α overexpression. F. Colony formation assay. G. CCK-8 assay. H. Analysis of the cell cycle distribution in ICCA cells upon TSA treatment and HP1α overexpression. I. RT-qPCR analysis of HP1α, STAT1, and ISGs expression upon TSA treatment and HP1α overexpression. J. Western blot analysis of HP1α and IFN signaling participants and proliferation and cell cycle-promoting factors after TSA treatment and HP1α overexpression. K. Western blot Analysis of the expression levels of intranuclear STAT1 and p-STAT1 upon TSA treatment and HP1α overexpression. L, M. Impact of HDAC1 knockdown on the expression of HP1α and IFN signaling L. RT-qPCR. M. Western blot. O-Q. Effect of HDAC1 knockdown on ICCA cell proliferation O. Cell cycle analysis. P. Colony formation assay. Q. CCK-8 assay. *P < 0.05. **P < 0.01
Fig. 5
Fig. 5
HP1α-HDAC1 complex regulates STAT1 expression in transcriptional level. A. The gel of the samples of control group and IP group. The sample was separated by SDS-PAGE and dyed by coomassie brilliant blue. The molecular weight of the protein ladder was shown. B, C. Evaluation of the interaction between HP1α and HDAC1. B. Exogenous co-IP. C. Endogenous co-IP. D. The scheme of the analysis for different domains of HP1α and HDAC1 protein. E. Mapping of the binding region of the HP1α-HDAC1 complex using exogenous co-IP. F. Evaluation of the interaction between HP1α and histone marks (H3K27ac and H3K9me3). G. Heatmap of the distribution of HP1α in the genome of HUCCT1 cells. The gradual change from red to blue represents the change in the number of peaks from most to least. H. Validation of the binding region of HP1α in STAT1 promoter by ChIP. I, J. Evaluation of the interaction between the HP1α-HDAC1 complex and the STAT1 promoter upon TSA treatment and functional domain deletion. I. HP1α. J. HDAC1. K. Evaluation of the distribution of histone marks in the STAT1 promoter upon HP1α knockdown using ChIP. L, M. Impact of HP1α-HDAC1 complex on the distribution of H3K27ac and H3K9me3. L. HP1α Knockdown and HDAC1 Overexpression. M. HDAC1 knockdown and HP1α overexpression. *P < 0.05. **P < 0.01
Fig. 6
Fig. 6
The proliferation of ICCA cells is inhibited by directly activating IFN signaling pathway. A. The effect of various concentrations of IFN-α2b on the proliferation of ICCA cells evaluated by CCK-8 assay. B. The heatmap of the RNA-seq data of IFN-α2b-treated ICCA cells. The gradual change from red to blue represents changes in gene expression from high to low. C. Western blot analysis of HP1α, IFN signaling participants and proliferation and cell cycle-promoting factors upon IFN-α2b treatment and HP1α overexpression. D. RT-qPCR analysis of the expression of HP1α, STAT1 and ISGs upon IFN-α2b treatment and HP1α overexpression. E. Analysis of the expression level of intranuclear STAT1 and p-STAT1 upon IFN-α2b treatment and HP1α overexpression by Western blot. F. The effect of IFN-α2b treatment and HP1α overexpression on the distribution of cell cycle of ICCA cells. G-I. The result of proliferation assay. G. Colony formation assay. H. CCK-8 assay over 24 h. I. CCK-8 assay within 1 h. J. RT-qPCR analysis of the expression of HP1α and IFN signaling participants at different point of time. K. Western blot analysis of the expression and the activation level of STAT1 at different point of time. L, M. Effect of drug treatment on the distribution of H3K9me3 and H3K27ac in the STAT1 promoter. L. TSA treatment. M. IFN-α2b treatment. *P < 0.05; **P��< 0.01
Fig. 7
Fig. 7
Broad-spectrum HDACi plus IFN preparation regimen maximizes the antiproliferation effects on ICCA. A, B. Evaluation of the proliferation inhibition induced by the TSA plus IFN-α2b regimen. (A) CCK-8 assay. (B) Colony formation assay. Blank, solvent for IFN-α2b, and murine IFN-α2. C. The distribution of cell cycle of ICCA cells. D. Western blot analysis of HP1α and proliferation and cell cycle-promoting factors. E. RT-qPCR analysis of the effect of TSA plus IFN-α2b regimen on ISG expression. Red, upregulated by TSA. Blue, downregulated by TSA. Green, failed to be significantly regulated by TSA. F. Presentation of liver lesions induced by hydrodynamic tail vein injection in the different treatment groups. Solvent, the recommended solvent for TSA in animal experiments. G. Scheme of the animal experiment. (1) Blank + Solvent; (2) IFN-α2 + Solvent; (3) IFN-α2 + TSA. One week after the hydrodynamic tail vein injection, different treatments were applied for two weeks. The survival status of mice was then assessed. Blue arrow, Solvent. Orange arrow, TSA. Green arrow, Blank. Red arrow, murine IFN-α2. H-K. Evaluation of the severity of tumorigenesis and the effect of the regimen. H. Ratio of liver weight to total weight. I. Ascites volume. J. ICCA lesions areas. K. Ki-67 positivity rate in the liver lesions. L. Survival analysis of the mice in different treatment groups. M. Presentation of the subcutaneous tumors. N, O. Volume (N) and Ki-67 positive rate of subcutaneous tumors (O) after different treatments. P. Graphic illustration of the HP1α-HDAC1-STAT1 regulatory axis in ICCA. STAT1 is directly and epigenetically repressed by the HP1α-HDAC1 complex. Treatment with TSA plus IFN significantly inhibited the proliferation of ICCA cells by targeting the HP1α-HDAC1 complex, increasing the total STAT1 level, downregulating HP1α, increasing basal IFN signaling, and directly stimulating the IFN pathway. *P < 0.05. **P < 0.01
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