Chromatin accessibility complex subunit 1 enhances tumor growth by regulating the oncogenic transcription of YAP in breast and cervical cancer

Cell line and transduction

HEK293T, MDA-MB-231 and Hela cells were purchased from Procell (Wuhan, China) and cultured in DMEM (Gibco) with 10% FBS (Gibco). For CHRAC1 knockdown, cancer cell was transduced with pLKO.1-puro (shNC) or pLKO.1-puro-shCHRAC1 (1# or 2#). Interfering sequences were: shCHRAC1-1#: 5′-ACTCCACTGTCTCTAAGTAAA-3′; shCHRAC1-2#: 5′-ACTCCACTGTCTCTAAGTAAA-3′.

Western blotting and Co-immunoprecipitation (Co-IP) assay

Cells were lysed in Beyotine lysis buffer (Cat#: P1003) containing protease inhibitor and phenylmethylsulfonyl fluoride (PMSF) at 4 °C for 20 min and centrifuged at 4 °C, 12000 rpm for 15 min. Then cell lysate was denatured by boiling with SDS sample buffer at 95 °C for 10 min and separated by 10% SDS-PAGE gel. After that, the membranes were incubated with specific primary antibody (ABclonal rabbit CHRAC1: A14896, 1:500; ABclonal rabbit CHRAC17: A6469, 1:1000) overnight at 4 °C, secondary antibody for 1 h at room temperature and analyzed with electrogenerated chemiluminescence (ECL) system (Cat#: WBKLS0500; Millipore, Burlington, MA, USA).

For Co-IP assays, cell lysates of 293T cells transfected with Flag-tagged CHARC1and HA-tagged YAP vectors were incubated with Agarose beads (Cat#: 16-266; Millipore, Burlington, MA, USA) and Flag antibody (E005; ABclonal) or IgG control overnight at 4 °C. The agarose beads/protein complex was washed 4 times with NP40 buffer and denatured at 95 °C for 15 min with SDS sample buffer and then subjected SDS-PAGE.

Bio-ID

Bio-ID experiment was conducted with reference to previous method (Zhang et al., 2018a). Briefly, cells that stably expressed YAP-BirA* were cultured in complete medium containing 50 µM of biotin for 24 h, then washed with pre-cooled 1 x PBS 6-8 times, lysed with 1ml lysis buffer including protease inhibitor, and sonicated. The supernatant was obtained by centrifugation (4 °C, 15,000 rpm, 10 min) and incubated with 50 µl Streptavidin-conjugated beads (17511301; GE Healthcare, Chicago, IL, USA) overnight at 4 °C. After washing with 2% sodium dodecyl sulfate (SDS), 1% Triton X-100, 250 mM LiCl, 50 mM Tris buffer and 50 mM NH₄HCO₃ buffer, the beads were stored at −20 °C for mass spectrometry or western blotting analysis. For western blotting detection, beads were denatured in 2X loading buffer at 100 °C for 10 min. Then supernatants were subjected SDS-PAGE. After blocking with 3% Bovine albumin (BSA) for 2 h at room temperature, the membranes were incubated with HRP-conjugated Streptavidin for 1 h at room temperature. After washing 4 times with TBST, the membranes were analyzed with electrogenerated chemiluminescence (ECL) system.

Survival analysis of CHRAC1

The Gene Expression Profiling Interactive Analysis version 2 (GEPIA2) (http://gepia2.cancer-pku.cn/) was used to investigate the overall survival (OS) and dis-ease-free survival (DFS) curves, as well as a survival map of CHRAC1 in The Cancer Genome Atlas (TCGA) tumor types. The expression threshold was set at 50% for high CHRAC1 expression and low CHRAC1 expression.

CCK-8, wound-healing and colony formation assays

Cell proliferation curves were measured by cell Counting Kit-8 (CCK-8) (BMU106-CN; Abbkine, Wuhan, China). Cells were seeded in 96-well plate with 4,000 cells in each well and cultured for 0, 24, 48, 72 and 96 h. Then the 450 nm absorbance was measured at each time point by adding CCK-8 reagent. For wound-healing assay, cells were inoculated in 6-well plates with 70% confluence and cultured for 36 h. Cell monolayer was scratched with the pipette tip to generate wound which was photographed every 12 h to monitor cell migration. For colony formation, cells were inoculated in 6-well plate with 1,000 (Hela) or 4,000 (MDA-MB-231) cells each well. After 10 days, the clones were stained with 0.05% crystal violet for imaging to monitor cell growth.

Real-time quantitative PCR (RT-qPCR)

RT-qPCR was used to verify the effect of CHRAC1 knockdown on YAP target genes in MDA-MB-231 and Hela cells. In this study, shNC was used as a control group and CHRAC1 shRNA (1# and 2#) was used as experimental group. Total RNA from cells was extracted according to the instructions of Vazyme RNA extraction Kit (Cat#: RC112-01; Vazyme, Beijing, China). RNA concentration and Purity were detected by Nanodrop spectrophotometer and 1µg RNA was applied to reverse transcription to synthesize complementary DNA (cDNA) using Reverse Transcription Kit (Cat#: RK20429, ABclonal) under the following conditions: 37 °C for 2 min, 55 °C for 15 min, 85 °C for 5 min. The reverse-transcribed cDNA was diluted 5-fold and RT-qPCR was conducted with the SYBR Green qPCR Mix reagent (Cat#: RK21203, ABclonal) on a Bio-Rad quantitative PCR instrument under the following conditions: 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference gene. The method to calculate the relative expression was 2^−ΔΔCt. All of the RT-qPCR reactions were performed in duplicates. RT-qPCR primers sequences are available in supplementary materials.

Immunofluorescence (IF)

Cells were cultured for 36 h, fixed with 4% paraformaldehyde, and blocked with Image-iT signal enhancer (Thermo: I36933). Thereafter, cells were incubated with primary antibody (Santa Cruz mouse YAP: sc-101199, 1:200; ABclonal rabbit CHRAC1: A14896, 1:200) overnight at 4 °C and secondary antibody for 1 h. For Ki67 staining, freshly dissected tumor tissue was fixed and embedded in OCT reagent (Sakura) and cut to 4µm sections. Thereafter, sections were treated with citrate buffer for antigen retrieval and the following steps are similar to cell immunofluorescence staining.

RNA-seq

After the extraction of total RNA, the sequencing library was generated using the Prep Kit V2 (Illumina, San Diego, CA, USA) for RNA Library according to the manufacturer’s procedure. RNA-seq library was sequenced using the 75-nucleotide paired-end sequencing protocol on a NextSeq sequencer (Illumina). The technical replicates are duplicates for each sample and the RNAseq details are as follows: sequencing depth: 34.10939x; dispersions: 0.017797; effect 2; false positive rate: 0.05. The statistical power of this experimental design, calculated in RNASeqPower is 0.98. For bioinformatics analysis: genes whose Fold Change (padj <= 0.05) is less than or equal to -2 were defined as down-regulated genes (CHRAC1 knockdown versus control cell). Heat-map for different cancer hallmarks was created by TB-tools. The sequencing data have been deposited in the FigShare (DOI: 10.6084/m9.figshare.23989101).

Mouse model

Five-week-old male and female BALB/c nude mice (60 single), there was a lack of mature T lymphocytes, were purchased from Animal Center in Tongji Medical College, and subsequently housed under specific-pathogen-free conditions (Temperature: 22  ± 2 °C, Relative humidity: 60  ± 10%, 12 h light/dark cycle) at the Animal Center in Tongji Medical College. All the mice were given an unrestricted access of sterile food and water. Nine single male/female mice were randomly assigned to three separate groups (each group: three singles (sample size is determined according to the results of the preliminary experiment)): shNC group (control group) and CHRAC1 shRNA group (1# and 2#) (experimental group). 5 × 10⁶ MDA-MB-231 or Hela cells treated with shNC or CHRAC1 shRNA were injected into the dorsal side of each nude mice (MDA-MB-231 cell: female mice; Hela cell: male mice). After about 2 weeks, tumor volume was monitored through double blind trials with vernier caliper every other day, and the size was measured as V = (L × W²) / 2 (L: length, W: width). Tumors were removed, imaged and weighed from mice with no significant weight change after 35 or 40 days. The tumor volume is generally no larger than 2000 mm³. The use and research design of animals was approved the Institutional Review Committee of Huazhong University of Science and Technology. The approval number: 2022 IACUC Number: 3148.

Immunohistochemistry (IHC) staining

Tissue sections of 48 human breast cancer and 48 cervical cancer specimens were purchased from Shanghai Wellbio Biotechnology company (Shanghai, China). Verbal informed consent was obtained and cancer specimens in this research have been approved by the Ethics Committee of of Huazhong University of Science and Technology and Shanghai Zhuoli Biotechnology Co., LTD (approval number: ZL2019-9-LL028, ZL2019-11-LL029). IHC staining was applied to detect the expression levels of CHRAC1 and YAP in breast and cervical cancer tissues. After deparaffinization and rehydration, antigen retrieval was performed. Endogenous peroxidase was blocked with 10% goat serum 1 h. Next, tissue microarrays were stained with primary antibodies (rabbit CHRAC1: Abclonal A14896, 1:200; rabbit YAP: Cell Signaling Technology 14074, 1:200) overnight at 4 °C and secondary antibody for 1 h. After washing, color development was performed with DAB kit (Maxim, DAB-0031) and the microarray was scanned with 3DHISTECH’s Slide Converter. The scanned images were opened with the Caseviewer software and enlarged to different magnifications. Click the “take snapshot” button on the software to take a picture. The images were saved in PNG format with 300 dpi resolution.

Statistical analysis

Statistical analysis was conducted with Prism 8 (GraphPad Software, La Jolla, CA, USA). Two-way analysis of variance (ANOVA) was used in CCK-8 and tumor volume detection assay. Student’s t-test (two-tailed) was performed to compare CHRAC1 expression, wound-healing ability, colony formation ability, tumor weight and Ki67 staining. Linear regression analysis was performed to investigate the correlation between CHRAC1 and YAP in cancer tissues.

Interactome of YAP and CHRAC1 is a potential Bio-ID interactor of YAP

To clarify YAP oncogenic regulators, we identified the spatial proximity interactome of YAP using the Bio-ID (proximity-dependent biotin identification) method (Fig. 1A). HEK293T cells expressing YAP-BirA*-HA were cultured in medium supplemented with or without biotin; much more biotinylated proteins were captured in total cell lysates cultured with biotin (Figs. 1B and 1C). Following mass spectrometry, we identified 254 potential interactors of YAP (YAP Bio-ID interactor). The Gene Ontology (GO) analysis on biological process for YAP Bio-ID interactors revealed a significant enrichment in Hippo pathway, cell division, cell–cell adhesion, DNA repair, DNA replication, mRNA processing and chromatin remodeling (Fig. 1D). In additional to numerous known YAP interactors (AMOT, LATS1 and AMOTL1), CHRAC1 attracted our attention. As a member of chromatin remodeling complex, CHRAC1 has been involved in transcription and DNA replication. However, the significance of CHRAC1 in cancer development has not been investigated extensively. Therefore, we focused on the study of CHRAC1.

High expression of CHRAC1 predicts advanced pathological tumor stages and poor survival

To explore the expression of CHRAC1 in cancer tissues, we analyzed the expression of CHRAC1 in cancer tissues including breast and cervical. Immunohistochemistry (IHC) result displayed that CHRAC1 was mainly expressed in the nucleus and the staining was extremely weak in para-tumor tissues, while breast and cervical cancer biopsies showed strong CHRAC1 signal (Figs. 2A and 2B).

To evaluate the potential clinical value of CHRAC1, we explored the correlation between CHRAC1 expression and patient survival within different tumors in the TCGA dataset (Fig. S1A). High CHRAC1 mRNA level was correlated with decreased overall and disease-free survival in cases of CESC and other tumor types by using the Gene Expression Profiling Interactive Analysis version 2 (GEPIA2) tool (Figs. 2C–2E, Figs. S1B–S1K). Additionally, the CHRAC1 level was significantly associated with pathological stages of kidney renal clear cell carcinoma (KIRC), adrenocortical carcinoma (ACC) and PAAD patients (Fig. S2). Moreover, in breast cancer specimens (n = 48), CHRAC1 expression increased with the progression of tumor stage (Figs. 2F and 2G). In cervical cancer specimens (n = 48), patients with metastasis had elevated CHRAC1 protein expression compared to those patients with primary tumors (Fig. 2H). Together, these data manifest that high CHRAC1 expression may predict advanced pathological tumor stages and poor survival in breast and cervical cancer.

Downregulation of CHRAC1 inhibits tumor growth

To validate the effect of CHRAC1 on cancer progression, we silenced CHRAC1 (Fig. 3A) in breast and cervical cancer cell lines (MDA-MB-231 and Hela). The downregulation of CHRAC1 have been confirmed by immunoblot, the result showed that the knock down of CHRAC1 specifically inhibited the protein level of CHRAC1 but not another family member CHRAC17 (Fig. S3A). CCK-8 assay displayed that CHRAC1 silencing suppressed the activity of MDA-MB-231 and Hela cells (Fig. 3B). Wound-healing assay also indicated that CHRAC1 down-regulation restrained cancer cell migration (Fig. 3C). In addition, colony formation assay showed that cancer cells with ablated CHRAC1 demonstrated significantly decreased colony size and number (Fig. 3D). To make the knockdown data more compelling, we also investigated the effect of CHRAC1 over-expression on cancer cells. The results showed that CHRAC1 over-expression significantly promoted cell migration and proliferation both in MDA-MB-231 and Hela cells (Figs. S3B–S3D). Collectively, these data suggest that CHRAC1 silencing inhibits the proliferation of breast and cervical cancer cells.

To verify the influence of CHRAC1 on tumorigenesis, we conducted a xenograft assay in nude mouse. Downregulation of CHRAC1 in Hela cells could significantly reduce tumor size, and tumor formation was not possible in MDA-MB-231 cells with CHRAC1 inhibition (Figs. 3E and 3F). The average weight of CHRAC1-silenced Hela xenografts was about one-third of the control group (Fig. 3G). To investigate the proliferation capacity of tumor cells, we performed immunofluorescence staining of Ki67 for tissue sections. The ratio of Ki67 positive cells was statistically reduced in CHRAC1-silenced tumors compared to control tumors (Fig. 3H). Collectively, these data display that CHRAC1 silencing suppresses tumor growth of breast and cervical cancer cells in nude mice.

Inhibition of CHRAC1 suppresses the oncogenic transcription of YAP

To illustrate the mechanism of CHRAC1 regulation on tumorigenesis, we performed RNA-seq assay in CHRAC1-silenced Hela cells. CHRAC1 ablation caused differential expression of 2,595 genes, of which 1,300 genes were increased and 1,295 genes were decreased (Fig. 4A). Gene ontology (GO) annotation of the 1,295 down-regulated genes indicated that they were highly enriched for GO terms linked to transcription, cell proliferation, cell apoptosis, cell migration and related signaling pathways (Fig. 4B). A heatmap of RNA-seq data clearly showed that knock-down of CHRAC1 suppressed the expression of representative cancer hallmarks, such as DNA repair, G2M checkpoint and the P53 pathway (Fig. 4C). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation displayed that CHRAC1 silencing resulted in significant downregulation (p < 0.01) of Hippo pathway-related genes (Fig. 4D). Then, we focused on the direct YAP target oncogenes previously identified (Zanconato et al., 2018; Zanconato et al., 2015). Gene set enrichment analysis (GSEA) displayed that YAP target gene signature was enriched in control Hela cells but not in CHRAC1 silenced cells, suggesting that CHRAC1 might trigger the oncogenic transcription program of YAP in Hela cells (Fig. 4E). Further, RT-qPCR results confirmed that depletion of CHRAC1 reduced the mRNA level of three classical YAP target genes (CTGF, CYR61, ANKRD1) (Fig. 4F). We also performed immunofluorescence (IF) assay for CTGF in Hela and MDA-MB-231 cell lines to validate our finding at the protein level. The results showed that knock down of CHRAC1 significantly decreased the fluorescence intensity of CTGF both in Hela and MDA-MB-231 cells (Fig. 4G). Together, these results demonstrate that CHRAC1 indeed influences the transcriptional activation of YAP.

CHRAC1 interacts with YAP and is positively correlated with YAP in breast and cervical cancer patients

According to the Bio-ID results, CHRAC1 may be a potential interactor of YAP. To validate the interaction of CHRAC1 and YAP, we performed exogenous co-immunoprecipitation (Co-IP) assay. The result showed that flag-tagged CHARC1 was able to specifically precipitated HA-tagged YAP in 293T cells compared to the IgG control (Fig. 5A, left panel). In addition, Co-IP detection was also conducted in CHRAC1 overexpressed MDA-MB-231 and Hela cells, and it was found that YAP could specifically co-purify CHRAC1 in both cells (Fig. 5A, right panel). Furthermore, immunofluorescence assay displayed that CHRAC1 co-localized with YAP both in MDA-MB-231 and Hela cells (Fig. 5B). Therefore, there is indeed an interaction between YAP and CHRAC1. Then, we investigated the clinical correlation of CHRAC1 and YAP and found CHRAC1 was associated with YAP across Pan-cancer, including BRCA and CESC in TCGA database (Figs. 5C–5E). In addition, both breast and cervical cancer biopsies with high CHRAC1 expression showed stronger YAP staining (Fig. 5F). Consistent with this, there was significant correlation ( p < 0.0001) between YAP and CHRAC1 in these cancer biopsies (Fig. 5F). Thus, CHRAC1 is elevated in breast and cervical cancer and the upregulation correlates well with YAP.

In summary, our study found that depletion of CHRAC1 suppresses cancer cell proliferation and tumor growth. The potential mechanism may be that CHRAC1 interacts with YAP to enhance the transcription of YAP down-stream oncogenes to promote tumor growth (Fig. 6). Moreover, CHRAC1 is frequently upregulated in diverse cancers and the upregulation is statistically associated with YAP activation and poor prognosis in cancer patients. These findings highlight CHRAC1 might be a promising candidate for cancer diagnosis and therapy.