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

**ong, Fei; Wang, Da; **ong, Wei; Wang, **n; Huang, Wen-hua; Wu, Guan-hua; Liu, Wen-zheng; Wang, Qi; Chen, Jun-sheng; Kuai, Yi-yang; Wang, Bing; Chen, Yong-jun

doi:10.1186/s13046-024-03070-3

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

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Published: 30 May 2024

Volume 43, article number 152, (2024)
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Unveiling the role of HP1α-HDAC1-STAT1 axis as a therapeutic target for HP1α-positive intrahepatic cholangiocarcinoma

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Fei **ong^1,2^na1,
Da Wang¹^na1,
Wei **ong³^na1,
**n Wang⁴,
Wen-hua Huang⁵,
Guan-hua Wu¹,
Wen-zheng Liu¹,
Qi Wang¹,
Jun-sheng Chen¹,
Yi-yang Kuai¹,
Bing Wang¹ &
…
Yong-jun Chen¹

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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.

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Introduction

Based on the anatomical location, cholangiocarcinoma is divided into four types: intrahepatic, perihilar, and extrahepatic cholangiocarcinoma (ICCA, PCCA and ECCA) and gallbladder cancer (GBC). ICCA is a heterogeneous group of malignant tumors comprising approximately 20% of all cholangiocarcinoma and is characterized by a high degree of malignancy and atypical early symptoms. The incidence of ICCA is relatively high in certain endemic areas with a higher incidence of inflammatory biliary diseases, and the incidence rate is increasing yearly [1]. Owing to the late-stage presentation, resistance to comprehensive treatments, and extremely high rate of postsurgical recurrence, the 5-year overall survival rate of ICCA is lower than 10% [2]. Therefore, the identification of novel molecular targets is critical.

Heterochromatin Protein 1α (HP1α, encoded by the CBX5 gene) is one of the most important HP1 family members and is a non-histone chromosomal protein involved in transcriptional silencing via heterochromatin formation and structural maintenance [3]. Many studies have shown that the distribution of HP1α on polytene chromosomes is not restricted to the chromocenters or telomeres. HP1α binds to chromatin mainly through direct interactions with modified histones, especially trimethylated H3K9 (H3K9me3), through the Chromo domain, and by interacting with other proteins through the Chromo shadow domain [4]. In cancerous lesions, HP1α is involved in the regulation of malignant behaviors, such as cell proliferation and cell cycle progression. Downregulation of HP1α has been demonstrated to inhibit the malignant biological behaviors of lung cancer, cervical cancer, and prostate cancer cells, for example, by impairing proliferation and inducing apoptosis [5,6,7]. Previously, we found that downregulation of Dicer and CyclinD1, which are binding proteins of HP1α, inhibited the proliferation of ICCA cells [8, 19,20]. Detailed information is provided in Table S1. The expression dataset of CCA in The Cancer Genome Atlas (TCGA) was downloaded from the UCSC Xena database. In this study, the fold-change values were obtained by logarithmic (log2FC) transformation. False-positive results were avoided by calculating of adjusted P values (adj. P) values using the Benjamini-Hochberg procedure. The differentially expressed genes (DEGs) were defined with the following cutoff values: adj. P < 0.05 and |log2FC| > 0.8. The tools and databases used for the bioinformatics analysis are summarized in Table S2 [21,22,23,24,25,26,27,28,29].

Cell culture

Human ICCA cell lines (HUCCT1, HCCC-9810, RBE, HUH28, and SSP-25) were maintained in our laboratory and cultured in RPMI-1640 medium. The kidney cell line, HEK-293T, was maintained in our laboratory and cultured in DMEM. All cell culture media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified incubator with 5% CO2. The mycoplasma was detected by colorimetric method with Mycolor One-Step Mycoplasma Detector (Vazyme, China) according to the manufacturer’s instructions.

Tissue microarrays, immunohistochemical (IHC) analysis and HE staining

The difference in HP1α expression between CCA and para-cancerous tissues was evaluated using a tissue microarray purchased from Outdo Biotech, China, containing 36 CCA samples and nine para-cancerous tissue samples. Additionally, 40 surgical ICCA and 40 paired para-cancerous tissues used for IHC and 90 surgical ICCA and 90 paired para-cancerous tissues used for real-time quantitative PCR (RT-qPCR) were collected from the Department of Biliary-Pancreatic Surgery, Tongji Hospital of Huazhong University of Science and Technology, Wuhan, China. The follow-up of these 90 ICCA patients was performed after surgery, and the date of death or last follow-up was recorded. All the research was conducted in accordance with both the Declarations of Helsinki and Istanbul. The experimental protocols were approved by the Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology (#TJ-IRB20230927). All patients signed informed consent forms.

The tissue samples were fixed with 4% polyoxymethylene and subjected to embedding and sectioning at 5 μm thickness. For HE staining, the sections were sequentially stained with hematoxylin and eosin. For IHC analysis, samples were deparaffinized, rehydrated and subjected to antigen retrieval. Then, the samples were blocked with 5% bovine serum albumin (BSA) prior to overnight incubation with primary antibodies at 4 °C (listed in Table S3). The next day, tissue samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies prior to DAB and hematoxylin staining. The intensity of staining (0, negative; 1, weak; 2, moderate; and 3, strong) and the percentage of positive cells (0, 0%; 1, 1–25%; 2, 26-50%; 3, 51–75%; and 4, 76-100%) were evaluated in a blinded manner. Finally, the IHC score was calculated (IHC score = staining intensity × percentage of positive cells).

Reagents and siRNA transfection

The inhibitors used in this study were as follows: Trichostatin A (TSA; HY-15144, 0.5 µM, 24 h), PF-06700841 (HY-112708, 50 nM, 24 h), decitabine (HY-A0004, 5 µM, 48 h), and GSK-J1 (HY-15648, 10 µM, 48 h) were purchased from MedChemExpress, China. Valproic acid (S1168, 1 µM, 48 h), Santacruzamate A (S7595, 20 µM, 48 h) and ACY-775 (S0864, 5 µM, 48 h) were purchased from Selleck, China. Human IFN-α2b (CYT-205, 60 ng/mL, 24 h) was purchased from Prospec (Israel). ICCA cells were transfected with siRNA (50 nM) and nonsense siRNA (50 nM) with Lipofectamine 2000 (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. The siRNA constructs were synthesized by Sangon Biotech (China), and all the sequences are listed in Table S4.

Plasmid construction and lentiviral transduction

Full-length and truncated sequences of Tripartite Motif Containing 28 (TRIM28), HDAC1 and CBX5, namely, full-length HDAC1 (Flag tagged, NM_004964.3), full-length CBX5 (HA tagged, NM_001127322.1), full-length TRIM28 (Flag tagged, NM_005762.3), truncated HDAC1 (lacking the histone deacetylase or disordered region) and truncated CBX5 (lacking the chromo domain or shadow domain), were generated by PCR and were inserted separately into the plasmid vector pHAGE-puro. Gene-specific small hairpin RNAs were synthesized by Sangon Biotech and were inserted into the pLKO.1-puro vector. HEK293T cells were transfected with the vectors mentioned above, along with the envelope plasmid pMD2.G and the packaging plasmid psPAX. The supernatant was filtered and collected after three days. ICCA cells were infected with the specific lentiviruses in the presence of HiTransG (1:25, GeneChem, China) for 24 h and then treated with 1 µg/ml puromycin to establish stable cell lines.

RNA extraction, RT‒qPCR and RNA sequencing (RNA-seq)

Total RNA from cell and tissue samples was extracted with RNA Extraction Reagent (Vazyme) and reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (+ gDNA Wiper) (Vazyme) according to the manufacturer’s instructions. RT-qPCR was performed using Hieff® qPCR SYBR Green Master Mix (No ROX, Yeasen, China) and specific primers (Table S4) in an iQ5™ quantitative PCR system (Bio-Rad, USA). Expression levels were calculated using the 2 ^− ΔΔCt method. Primers were synthesized by Sangon Biotech.

Total RNA samples were used for RNA-seq performed by HaploX (China). Quality was evaluated using a 4200 TapeStation system (Agilent, USA) and quantified using a Life Invitrogen Qubit 3.0 fluorometer (Thermo Fisher Scientific). The samples were used for library preparation, followed by RNA-seq by Illumina PE150 sequencing (Illumina, USA). The raw data were used for further bioinformatics analysis.

Western blot

Fresh cell samples were lysed with RIPA buffer in the presence of proteinase inhibitor cocktail and PhosSTOP phosphatase inhibitor (Roche, Switzerland). Total protein was extracted, mixed with 5× loading buffer, and denatured at 95 °C for 5 min. Proteins in individual lysate samples (30 µg per lane) were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, USA). Then, the membranes were blocked with 5% BSA and incubated with primary antibodies overnight at 4 °C. The next day, the membranes were incubated with secondary antibodies, and the signals were visualized using enhanced chemiluminescence (Thermo Fisher Scientific). The results were analyzed using Image Lab software (Bio-Rad).

Purification and isolation of nuclear and cytoplasmic proteins

Nuclear and cytoplasmic proteins were purified and isolated with Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). Briefly, ICCA cells were mixed with cytoplasmic extraction buffer A followed by vigorous vortexing and incubation in an ice bath. Cytoplasmic extraction buffer B was then added, followed by vigorous vortexing and centrifugation. The supernatant was collected as a cytoplasmic sample. The precipitate was mixed with nuclear extraction buffer, followed by vigorous vortexing, incubation in an ice bath, and centrifugation. The supernatant was collected as a nuclear sample for further analysis.

Coimmunoprecipitation (co-IP) and Electrospray Ionization Mass Spectrometry (ESI-MS)

For the exogenous co-IP assay, HEK293T cells were transfected with expression vectors carrying full-length or truncated coding sequences. For the endogenous co-IP assay, wild-type ICCA cells were cultured without any specific treatment. The cells were harvested and lysed with IP buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 1% NP-40) in the presence of proteinase inhibitor cocktail and PhosSTOP. The samples were subjected to ultrasonication and centrifugation. 5% of the supernatant was collected as the input sample, and the remaining supernatant was incubated with primary antibodies and protein A + G agarose beads (Med Chem Express). The next day, the agarose beads were washed with IP buffer and heated to 95 °C in 2× loading buffer as the IP sample. All the protein samples were used for western blotting analysis. The IP samples of HEK293T cells transfected with the HP1α expression vectors and control vectors were used for ESI-MS analysis. The analysis was performed by Applied Protein Technology (China).

Enzyme linked Immunosorbent Assay (ELISA)

The supernatants of ICCA cells were collected without dilution. The concentrations of IFN-α and IFN-γ were evaluated using a Human IFNA1 ELISA Kit (KE00044, Proteintech, China) and a Human IFN-gamma ELISA Kit (RK00015, ABclonal, China) according to the manufacturer’s instructions. The supernatant and the reaction mixture were incubated at 37 °C, and the absorbance at 450 nm was measured using a MULTISKAN FC microplate reader (Bio-Rad).

Cleavage under targets and tagmentation (CUT & tag)

A Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme) was used to evaluate the genomic occupancy of DNA binding proteins. Briefly, 1 × 10⁵ ICCA cells were collected and the nuclei were extracted. Specific DNA fragments were obtained as previously described [Full size image

We failed to detect binding between HP1α and STAT1 (Figure S3C); therefore, we explored the interaction between HP1α and the STAT1 promoter region. We failed to detect any difference in luciferase activity upon HP1α overexpression, although insertion of the promoter region (1000 bp to the TSS) significantly enhanced transcription (Figure S3D). Similarly, TSA application failed to influence the transcriptional activity mediated by the STAT1 promoter, which is inconsistent with the observations in ICCA cells (Figure S3E), indicating that indirect binding to chromatin, for example, interactions via histone modifications, should be examined. Although an interaction between HP1α and H3K9me3 was observed, no binding to H3K27ac or H3K9ac was observed (Fig. 4F and S3F). To explore the distribution in local chromatin, we calculated the number of sites in each section. Most of the sites were located in the 700–1000 bp region (Figure S3G). CUT & Tag was performed to evaluate the occupancy of HP1α and histone markers (Fig. 5G, S3H). The binding sequences of the different markers are summarized in Table S8. The sequence of the HP1α binding sites was similar to that of H3K9me3, rather than acetylated histones. Most peaks in the promoters were located upstream of the 1000 bp. Except for H3K27me3, all targets exhibited peaks in the promoter region of STAT1, which was similar to the results obtained in the GEO database (Figure S3I, S3J). To extend this conclusion, we screened for all genes that contained four peaks of histone marks in the same regions (9063 genes, Figure S3K, S3L). Among these 9063 genes, 5871 contained HP1α peaks, and some genes were involved in cell cycle regulation and viral infection (Figure S3M). Among these 5871 genes, 1157 genes were significantly regulated by TSA and were highly correlated with cell cycle regulation (Figure S3N).

HP1α was found to be significantly enriched in the 700–1000 bp region of the STAT1 promoter compared to other proximal sites (Fig. 5H). The binding of HP1α to this region was inhibited by deletion of the Chromo domain or by treatment with TSA (Fig. 5I). Similarly, HDAC1 could bind to this region, and this binding was inhibited by deletion of the Histone Deacetylase domain or treatment with TSA (Fig. 5J). Additionally, only increases in H3K27ac and decreases in H3K9me3 were observed in this region after HP1α knockdown (Fig. 5K). Both H3K27ac and H3K9me3 were found to be enriched in the promoters of certain genes involved in infection and cell cycle (Figure S4A, S4B). ChIP was performed to evaluate the effect of the HP1α-HDAC1 complex on the local distribution of the two histone markers. Once either HP1α or HDAC1 was knocked down, we observed higher levels of H3K27ac and lower levels of H3K9me3, which could be partly rescued by overexpression of the other parts (Fig. 5L and M). Considering this evidence, we suggest that the HP1α-HDAC1 complex regulates the basal activation of IFN signaling by transcriptionally downregulating STAT1.

The proliferation of ICCA cells was inhibited by directly activating IFN signaling pathway

To evaluate the effect of the IFN signaling pathway, IFN-α2b, a cytokine preparation that can directly and potently activate IFN signaling, was used. We attempted to determine the optimal concentration, and 60 ng/mL was selected because higher concentrations had limited inhibitory effects (Fig. 6A). HUCCT1 cells were treated and RNA-seq was performed. We found that certain ISGs were upregulated (Fig. 6B). Among the 5871 genes mentioned above, 161 were regulated by IFN-α2b, some of which were involved in IFN signaling and translation initiation (Figure S3O).

IFN-α2b significantly induced the expression of STAT1, p-STAT1, ISG15, SAMHD1, and MX1 and downregulated the protein factors (Fig. 6C and D). HP1α is not regulated by IFN-α2b. IFN-α2b treatment induced synchronous upregulation of intranuclear STAT1 and p-STAT1 (Fig. 6E). IFN-α2b inhibited proliferation and induced S-phase arrest (Fig. 6F and H, S3P). HP1α overexpression partially reversed these effects. Notably, an inhibitory effect was observed as early as 24 h after treatment (Fig. 6H). In contrast, no similar effect was observed within 1 h (Fig. 6I). To investigate this possibility, we examined the expression levels of the related molecules at different time points. Although all of these factors, except for STAT1, were upregulated before the 1 h time point, their expression was upregulated more drastically at 24 h and gradually decreased thereafter (Fig. 6J). However, HP1α expression was not affected. The protein levels of p-STAT1, but not total STAT1, increased within 1 h. Up to 24 h, the levels of both total STAT1 and p-STAT1 were elevated (Fig. 6K). IFN-α2b and TSA induced a significant increase in H3K27ac levels in the STAT1 promoter. TSA also decreased the local H3K9me3 levels (Fig. 6L and M). It could be inferred that increased total STAT1, rather than increased p-STAT1, is the underlying cause of proliferation inhibition, which is consistent with the finding that the HP1α-STAT1 axis regulates ICCA cell proliferation (Fig. 3).

Broad-spectrum HDACi plus IFN preparation regimen improves the antiproliferation effects and inhibits the development of ICCA

Proliferation assays were performed to evaluate the effects of broad-spectrum HDACi and IFN preparations. Compared with IFN-α2b alone, TSA plus IFN-α2b further inhibited the proliferation of ICCA cells (Fig. 7A and B, S4C). When TSA was applied with IFN-α2b-treated cells, we observed G1/G0-phase arrest, which was similar to the recruitment effect of chemotherapy regimens (Fig. 7C). TSA plus IFN-α2b further downregulated proliferation and cell cycle factors (Fig. 7D).

Given that both reagents could induce IFN signaling, we screened for common targets using RNA-seq data of both TSA and IFN-α2b, for example, ISGs (Figure S4D, Table S9). As previously reported [37], TSA abrogated IFN-α2b-mediated upregulation of some ISGs that were either downregulated or unaffected by TSA (Fig. 7E). STAT1 and three ISGs, including SAMHD1 and ISG15, were further upregulated by TSA, which was consistent with the RNA-seq results. The upregulation of some ISGs (including MX1) was not reversed by TSA treatment (Fig. 6E). CD274 and PDCD1LG2 (encoding PD-L1 and PD-L2, respectively) were used as the positive controls as both genes were upregulated upon IFN stimulations [38]. HP1α knockdown downregulated the mRNA level of CD274 and PDCD1LG2 (Figure S4E). IFN-α2b induced the transcription of both genes, which was slightly rescued by TSA treatment (Figure S4F). The mRNA level of CD274 was positively correlated with that of HP1α in ICCA samples (Figure S4G). When HP1α was knocked down, CD274 was then downregulated (Figure S4H). IFN-α2b upregulated the expression of PD-L1, while TSA downregulated PD-L1 whether IFN-α2b was applied or not (Figure S4H).

This combination has been validated in a mouse model. We established ICCA models and found that although IFN-α2 alone ameliorated the progression of ICCA, the additional application of TSA further inhibited this oncogenic process. This therapeutic effect was abrogated by Hp1α overexpression (Fig. 7F and G, and S4I). Neither the TSA plus IFN-α2 regimen nor IFN-α2 monotherapy led to morphological changes in the kidneys (Figure S4J). All ICCA lesions were confirmed to be of an epithelial origin (Figure S4K). The TSA plus IFN-α2 regimen further reduced the weight and area of hepatic lesions and the rate of Ki-67 positivity, yet the volume of ascites was not influenced. Hp1α-overexpressing tumors were more severe and insensitive to TSA and IFN-α2 (Fig. 7H and K, S4L). Murine Cd274 was significantly upregulated upon Hp1α overexpression, which was similar to the findings in ICCA samples (Figure S4M). In addition, the TSA plus IFN-α2 regimen further improved the prognosis of the mouse model, which was not observed in the Hp1α overexpression group (Fig. 7L). Additionally, TSA plus IFN-α2 further reduced the volume of subcutaneous tumors and the rate of Ki-67 positivity (Fig. 7M and O, S4N).

In conclusion, the following mechanism is proposed. Although minimal IFN signaling is maintained, STAT1 is repressed by the HP1α-HDAC1 complex in a direct and epigenetic manner. Treatment with broad-spectrum HDACi plus IFN significantly inhibited the proliferation of ICCA cells by increasing the total STAT1 level, downregulating HP1α, increasing basal IFN signaling, and directly stimulating the IFN pathway (Fig. 7P).

Discussion

The onset of ICCA is a global public health problem, and its incidence is increasing, particularly in regions with a high incidence of inflammatory lesions. Therefore, targeting these inflammation-related molecular mechanisms may be a therapeutic option for ICCA, although little is known about the relationship between ICCA and inflammatory signaling. In this study, we demonstrated the role of the HP1α-HDAC1-STAT1 axis as a potential target for activating the IFN signaling pathway and inhibiting ICCA cell proliferation.

As important nonhistone chromosomal protein, HP1α has been found to be associated with proper mitosis, cell cycle progression and DNA repair in multiple species and in various tissue types [3]. While heterochromatin markers are often increased in cancerous lesions [39], HP1α is upregulated in breast cancer [7]. In lung cancer, HP1α downregulation impairs cell viability by inhibiting the Wnt signaling pathway [5]. In cervical cancer, HP1α downregulation correlates with aberrant mitosis [7]. In prostate cancer, HP1α knockdown significantly induces apoptosis and growth arrest [6]. Similarly, HP1α is upregulated in ICCA. HP1α regulates proliferation, but not apoptosis or invasion, of ICCA cells. Notably, HP1α is downregulated in thyroid cancer, especially in metastatic and poorly differentiated lesions [40]. Therefore, the carcinogenic function of HP1α may be conserved in certain types of cancer.

Although the relationship between HP1α and IFN signaling remains unclear, supporting evidence has been reported. In medulloblastoma, HP1α silenced apoptosis-related inflammatory response genes. Another family member, CBX4, influenced retroviral genomic latency [41, 42]. We found that HP1α downregulation stimulated basal IFN signaling, which plays essential roles in impairing excessive proliferation via a mechanism based on ISG induction, STAT1 upregulation, and STAT1 nuclear translocation [10]. Additionally, we demonstrated that STAT1 is the central node in the crosstalk between HP1α and IFN signaling, based on the following observations. First, HP1α failed to regulate autocrine IFN and related signaling pathways. Second, the function of HP1α was unidirectionally regulated by the activated upstream kinase and blocked by IRF9 knockdown and downstream ISGF3 elimination. Third, intranuclear STAT1 and p-STAT1 were both synchronously upregulated upon HP1α knockdown. Fourth, HP1α expression remained stable upon the application of the treatments described above. The HP1α-STAT1 axis was further validated to inhibit the proliferation of ICCA cells in vivo and in vitro via the induction of IFN signaling and upregulation of ISGs, which seemed to conflict with the consequence of stemness maintenance arising from the passive regulation of STAT1 and IFN signaling in breast cancer [43, 44].

Next, we demonstrated that STAT1 might be regulated by histone acetylation using online database analysis, TSA treatment assays, and RNA-seq analysis. This conclusion was further validated in studies using cell lines derived from other tissues. Rampazzo et al. noted that TSA inhibited the IFN pathway in glioblastoma cells [45]. We observed the downregulation of some ISGs in the IFN pathway, which could be partially explained by differences among tissues [34]. However, we hypothesized that IFN signaling was activated in ICCA cells, despite its limited level, because TSA upregulated another group of critical genes, STAT1 and STAT2. In line with this conclusion, we found that STAT1 was regulated by HDAC1, but not by HDAC2 or HDAC6, by siRNA transfection or specific inhibitor treatment. HDACs can catalyze the removal of acetyl groups from lysine residues in the amino-terminal region of histone H3 and sometimes function as oncogenes [34, 37, 44]. In view of the common functions of DNA binding and increasing chromosome density, the interaction between HP1α and HDAC1 in the five HDACs in the database was verified in ICCA cells using co-IP and ESI-MS. This interaction was reported casually by Hauri et al., Li et al., and Zhang et al., but the binding pattern has not been discussed in detail [46,47,48]. Although crosstalk between HDACs and STAT1 has been reported previously, most studies have focused on the activation status rather than the transcriptional level of STAT1 [49, 50]. The HP1α-HDAC1 complex was located in the STAT1 promoter region in a H3K9me3-dependent manner, and this location was validated using CUT&Tag, co-IP, and ChIP. Compared to functional domain deletion, TSA application only partially abrogated this interaction in the local environment and increased the basal intensity of IFN signaling.

Unlike basal activation by TSA, direct activation of the IFN pathway by IFN-α2b drastically induced STAT1 and ISG expression in a time-dependent manner. Both activation mechanisms significantly inhibited the proliferation of ICCA cells, which was rescued by HP1α overexpression, indicating a role for HP1α in TSA and IFN-α2b resistance. This phenomenon may be explained by transcriptional regulation. We found that many genes, including STAT1, contained multiple histone marks and were occupied by HP1α. Some of these genes are involved in antiproliferative and antimicrobial processes. These are critical factors in the IFN pathway. TSA-regulated genes influence cell proliferation. IFN-α2b-regulated genes tend to influence antimicrobial function. Notably, we identified many terms related to protein translation initiation that were associated with the regulation of protein expression. This might explain why no proliferation-related terms were enriched in the RNA-seq data, even though the protein factors were significantly differentially regulated. Hansen et al. demonstrated that methylated H3K9 can impair IFN signaling activation in acute myeloid leukemia [51]. We found that HP1α knockdown affected the local distribution of histone modifications; for example, more H3K27ac and less H3K9me3, although the overall level of H3K27ac was stable. TSA exerted similar effects, whereas IFN-α2b increased only H3K27ac levels. TSA significantly downregulated HP1α expression, suggesting a potential strategy for the treatment of HP1α-positive ICCA. Broad-spectrum HDACi could not be replaced by specific HDACi for the limited anti-proliferative effects.

In this study, we evaluated the treatment effect of a broad-spectrum HDACi plus IFN preparation regimen and found that this combination maximized the antiproliferative effect. This regimen has been shown to be promising in two distinct murine models. This combination did not result in a visible injury to the kidneys. HP1α overexpression enhances resistance to this regimen. Another consequence of this regimen is regulation of IFN-related genes. Both PD-L1 and PD-L2 are upregulated upon IFN treatment, which could be regarded as a side effect of IFN preparations [10]. TSA treatment slightly reversed this effect. Similarly, the combination of IFN-α2b and TSA downregulated some ISGs, particularly those that were not elevated by TSA monotherapy, as previously reported in acute myeloid leukemia [37]. However, we believe that this contradiction can be partially explained by the high-throughput assessment. TSA treatment also increased the expression of some ISGs (represented by SAMHD1). TSA occasionally failed to overcome IFN-α2b stimulation (represented by MX1). Therefore, TSA seemed to have little effect on the antitumor ISG profile of IFN-α2b based on the finding that proliferation was significantly impaired by this combination, which supports its application.

Our study has several advantages. First, we detected only the HP1α-HDAC1 complex in ICCA cells, although other HDACs can also bind to other tumors. Second, we elucidated the relationship between HP1α and antitumor IFN signaling. STAT1 was identified as the central node and is regulated by the HP1α-HDAC1 complex. Third, to better utilize this molecular mechanism (a common biological function, a common function in regulating histone marks, and the combination of basal and direct activation), we validated the effect of the IFN-α2b plus TSA regimen. However, this method also has several disadvantages. First, this study was limited by the relatively low incidence of ICCA; therefore, multicenter ICCA cohorts were not obtained. Second, the detailed molecular mechanism underlying the regulation of each ISG, especially MX1, remains unclear, although the induction of additional ISGs could be interpreted by the molecular mechanism we found. Third, although the regimen in this study was proven to be promising, it was not validated in ICCA patients, which requires future work and more evidence.

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, which requires more evidence and clinical trials.

Data availability

The datasets downloaded, generated, and analyzed during the present study are available from the corresponding author upon reasonable request. Raw RNA-seq and CUT & Tag data were uploaded to the GEO database (GSE252094, GSE252095, GSE252096, and GSE252098).

Abbreviations

CCA:: Cholangiocarcinoma
ICCA:: Intrahepatic cholangiocarcinoma
HCCA:: Hilar cholangiocarcinoma
ECCA:: Extrahepatic cholangiocarcinoma
GBC:: Gallbladder cancer
HP1α:: Heterochromatin Protein 1α
H3K9me3:: Trimethylated H3K9
IFN:: Interferon
PARP:: Poly (ADP-ribose) polymerase
IFN-I, II, III:: Type I, II and III interferon signaling
FDA:: The Food and Drug Administration
STAT1:: Signal Transducer and Activator of Transcription 1
HDACi:: Histone deacetylase inhibitor
SPF:: Specific pathogen-free
MX1:: MX dynamin-like GTPase 1
ISG:: IFN-stimulated gene
SAMHD1:: SAM domain and HD domain 1
JAK1:: Janus kinase 1
TYK2:: Tyrosine kinase 2
TIC:: Transcription initiation complex
ISGF3:: IFN‐stimulated Gene Factor 3
H3K27ac:: Acetylated H3K27
HDAC:: Histone deacetylase
CHAF1A:: Chromatin Assembly Factor 1 Subunit A
POGZ:: Pogo Transposable Element with ZNF Domain
TRIM28:: Tripartite Motif Containing 28
p-STAT1:: Phosphorylated STAT1

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Acknowledgements

Not Applicable.

Funding

This study was supported in part by grants from the National Natural Science Foundation of China (#81974438, #82173069, and #81502108). The authors declare that there is no financial relationship with the organizations that sponsored this study, and the funding body did not participate in the study design, data collection, analysis, or writing of this paper.

Author information

Fei **ong, Da Wang and Wei **ong contributed equally to this work.

Authors and Affiliations

Department of Biliary‑Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.1095 Jiefang Road, Wuhan, Hubei, 430074, China
Fei **ong, Da Wang, Guan-hua Wu, Wen-zheng Liu, Qi Wang, Jun-sheng Chen, Yi-yang Kuai, Bing Wang & Yong-jun Chen
Department of General Surgery, Bei**g Friendship Hospital, Capital Medical University Bei**g, Bei**g, 100050, China
Fei **ong
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China
Wei **ong
Departement of Pediatric Surgery, Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, 430016, China
**n Wang
Department of Emergency, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China
Wen-hua Huang

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Fei **ong
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Da Wang
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Wei **ong
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**n Wang
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Wen-hua Huang
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Guan-hua Wu
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Wen-zheng Liu
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Qi Wang
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Jun-sheng Chen
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Yi-yang Kuai
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Bing Wang
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Yong-jun Chen
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Contributions

Y.C. and B.W. designed most of the experiments. F.X., D.W., and W.X. performed the experiment. F.X. wrote the manuscript. W.H., X.W., G.W., W.L., Q.W., J.C., Y.K. interpreted and analyzed the data. Y.C. and B.W. checked the manuscript. All of the authors have read and approved the manuscript.

Corresponding authors

Correspondence to Bing Wang or Yong-jun Chen.

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Ethics approval and consent to participate

All the research was conducted in accordance with both the Declarations of Helsinki and Istanbul. The experimental protocols were approved by the Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology (#TJ-IRB20230927). All patients signed informed consent forms. The animal experiments were approved by the Animal Experimentation Ethics Committee of Tongji Medical College (#TJH-202208006). Animal care and experimental procedures were performed according to the criteria outlined in NIH guidelines. The maximal tumor size permitted by their ethics committee was 1000 mm³. All the tumor lesions in this study were below 1000 mm³. All the animals were housed in a specific pathogen-free environment, including bedding, caging systems, and diet.

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The original online version of this article was revised: Affiliation 1 should be added to author, Fei **ong’s affiliation details.

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**ong, F., Wang, D., **ong, W. et al. Unveiling the role of HP1α-HDAC1-STAT1 axis as a therapeutic target for HP1α-positive intrahepatic cholangiocarcinoma. J Exp Clin Cancer Res 43, 152 (2024). https://doi.org/10.1186/s13046-024-03070-3

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Received: 14 January 2024
Accepted: 17 May 2024
Published: 30 May 2024
DOI: https://doi.org/10.1186/s13046-024-03070-3

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

Abstract

Background

Methods

Results

Conclusions

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Introduction

Cell culture

Tissue microarrays, immunohistochemical (IHC) analysis and HE staining

Reagents and siRNA transfection

Plasmid construction and lentiviral transduction

RNA extraction, RT‒qPCR and RNA sequencing (RNA-seq)

Western blot

Purification and isolation of nuclear and cytoplasmic proteins

Coimmunoprecipitation (co-IP) and Electrospray Ionization Mass Spectrometry (ESI-MS)

Enzyme linked Immunosorbent Assay (ELISA)

Cleavage under targets and tagmentation (CUT & tag)

The proliferation of ICCA cells was inhibited by directly activating IFN signaling pathway

Broad-spectrum HDACi plus IFN preparation regimen improves the antiproliferation effects and inhibits the development of ICCA

Discussion

Conclusions

Data availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Ethics approval and consent to participate

Consent for publication

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Electronic supplementary material

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