Abstract
Background
Ovarian clear cell carcinoma (OCCC) is an aggressive epithelial subtype characterized by intrinsic chemoresistance and limited targeted therapies. Transcriptional programs driven by Yes-associated protein (YAP) and TEA domain (TEAD) factors support tumor stress adaptation, suggesting that this axis represents therapeutic vulnerability. Histone deacetylase 6 (HDAC6) has been linked to Hippo/YAP regulation. We hypothesized that dual inhibition of HDAC6 and YAP-TEAD would converge on the YAP axis and yield synergistic cytotoxicity in OCCC.
Methods
We first analyzed a tissue microarray (TMA) of 171 OCCC cases stained for HDAC6, YAP, and connective tissue growth factor (CTGF) to assess correlations and survival associations. Guided by these findings, we treated HAC2, OVTOKO, and RMG-1 cells with fixed-ratio combinations of the HDAC6 inhibitor citarinostat and the YAP-TEAD disruptor verteporfin (VP). Drug interactions were quantified using Loewe isobolograms and the Chou-Talalay combination index (CI). Immunofluorescence was used to measure total YAP (t-YAP) and the nuclear-cytoplasmic (N/C) ratio, while RT-qPCR assessed YAP, CTGF, and CYR61 transcripts. In HAC2, western blotting analysis was performed for the expression of CTGF protein. The selected assays were reproduced with the distinct YAP-TEAD inhibitor CA-3.
Results
In patient tissues, cytoplasmic HDAC6 correlated positively with cytoplasmic YAP and CTGF, and inversely with nuclear HDAC6, but none of the markers predicted survival. Across all three cell lines, dual treatment produced robust, high-effect synergy (minimum CI ≈ 0.08–0.28). In HAC2, the combination increased t-YAP but reduced the N/C ratio, indicating restricted nuclear access. VP alone decreased t-YAP, whereas synergy was confirmed with CA-3, suggesting that TEAD disruption is the relevant mechanism. RT-qPCR revealed divergent cell line-specific transcriptional responses, with CTGF and CYR61 often upregulated by citarinostat and suppressed by VP or combination. The CTGF-HDAC6 correlation in the TMA supports the biological relevance of this axis, whereas early transcript changes likely reflect compensatory feedback.
Conclusions
An investigation from the clinic to bench identified an HDAC6-YAP-CTGF axis in OCCC and demonstrated that dual HDAC6 and YAP-TEAD inhibition yields strong high-effect synergy by restricting YAP nuclear access and limiting TEAD engagement. These findings may provide a rationale for biomarker-guided validation of citarinostat-VP combinations in organoids, in vivo models, and early-phase clinical studies.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12935-026-04204-7.
Introduction
Ovarian clear cell carcinoma (OCCC) is an aggressive epithelial ovarian cancer subtype characterized by intrinsic chemoresistance and a lack of effective strategies [1, 2]. Standard platinum-based regimens are often insufficient and are required for effective combination strategies that disable core survival programs rather than single agents. Yes-associated protein (YAP) and the TEA domain (TEAD) are related to tumor progression, including proliferation and therapy tolerance across solid tumors [3–7]. Similarly, in OCCC, YAP is related to tolerance to therapy and a poor prognosis [8, 9]. However, the precise regulatory network that maintains YAP activation in OCCC remains insufficiently defined.
Histone deacetylase 6 (HDAC6) is a cytosolic deacetylase for non-histone substrates and is reported to be a modulator of Hippo/YAP expression [10–12]. HDAC6 deacetylates and activates components of the Hippo pathway, including the mammalian STE20-like protein kinase 1 (MST1) [11] and MOB kinase activator 1 (MOB1) [10]. This activity has the potential to relieve Hippo restraint and favor YAP activation. Consistently, pharmacologic HDAC6 inhibition has been associated with reduced YAP protein abundance and altered nuclear-cytoplasmic distribution [10–12]. These findings suggest a potential HDAC6–YAP signaling axis that could represent a therapeutic vulnerability in OCCC.
Despite these insights, the clinical relevance and therapeutic potential of concurrently targeting HDAC6 and YAP–TEAD remain unexplored in OCCC. Previous studies have approached these pathways independently, leaving unclear whether dual inhibition can achieve synergistic effects sufficient to overcome intrinsic chemoresistance. Because both citarinostat (an HDAC6-selective inhibitor) and verteporfin (a YAP-TEAD inhibitor) are clinically applicable compounds, this combinatorial strategy holds promise for rapid translational development [13–15].
On this basis, we first evaluated a large OCCC tissue microarray (TMA) to define the clinical and molecular relationships among HDAC6, YAP, and the YAP target connective tissue growth factor (CTGF). We then examined the biological impact and mechanistic basis of dual HDAC6 and YAP–TEAD inhibition in OCCC cell models. This study, linking clinical pathology to experimental validation, aims to provide a new framework for targeting the HDAC6-YAP axis as a translationally actionable vulnerability in OCCC.
Materials and methods
Immunohistochemical staining
A total of 171 OCCC tissue microarray (TMA) sections were stained for HDAC6 (rabbit polyclonal, 1:250; Abcam, cat. #ab1440), YAP (rabbit monoclonal, 1:250; Cell Signaling Technology, cat. #14074), and CTGF (rabbit polyclonal, 1:1000; Proteintech, cat. #25474-1-AP). The staining intensity was semiquantitatively scored using a four-tier system: negative (0), ± (1), + (2), and ++ (3). Prior to scoring, representative examples for each category were selected to calibrate the criteria (Fig. 1, Figure S1). Two independent pathologists, blinded to clinical data, then scored all the cases according to these predetermined criteria. Correlations between markers were evaluated via Spearman’s rank correlation coefficient. Kaplan-Meier survival curves were generated for overall survival (OS) and progression-free survival (PFS) in relation to these molecules. Prognostic analyses were performed using univariate Cox proportional hazards models. Statistical analyses were carried out with IBM SPSS Statistics v25.0 (IBM Corp., Armonk, NY, USA). A two-sided p value < 0.05 was considered statistically significant.
Fig. 1.
Immunohistochemical expression of YAP, HDAC6 and CTGF. (A-C) HDAC6: (A) negative [no nuclear or cytoplasmic staining; score 0 (-)], (B) cytoplasmic positive [c-HDAC6; score 3 (++)], (C) nuclear positive [n-HDAC6; score 3 (++)]. (D-F) YAP: (D) negative [no nuclear or cytoplasmic staining; score 0 (-)], (E) cytoplasmic positive [c-YAP; score 3 (++)], (F) nuclear positive [n-YAP; score 3 (++)]. (G-H) CTGF: (G) negative [score 0 (-)], (H) positive [score 3 (++)]. Scale bars, 50 μm. Representative examples used for score calibration are provided in Figure S1
Cell lines and cell culture
HAC2 (AT-rich interactive domain-containing protein 1 A [ARID1A]-wild type) and OVTOKO (ARID1A-loss) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin). RMG-1 (ARID1A-wild type) was cultured in Ham’s F-12 Nutrient Mixture (F-12) supplemented with the same supplements. The cells were maintained at 37 °C in a humidified incubator with 5% CO₂.
Compounds and dosing design
Citarinostat (MedChemExpress, cat. #HY-15994) and VP (Selleck, cat. #S1786) were dissolved in DMSO to prepare aliquoted stocks. Combinations were administered at fixed ratios (1:1, 1:2, 2:1, and citarinostat: VP) spanning IC50 multiples, covering 0.06× to 4× the single-agent IC50 estimated per plate. VP was handled under light-protected conditions. For viability, the cells were treated for 48 h, and for IF/qPCR, they were treated for 24 h.
Cell viability and drug-interaction analysis
Cell viability was measured with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) (Roche). OCCC cells (5,000 cells/well, 96-well) were seeded overnight and treated for 48 h with citarinostat, light-protected VP or fixed-ratio combinations (1:1, 1:2, 2:1) across IC50-spanning doses. The absorbance was read at 550 nm with 650 nm reference (Multiskan SkyHigh, Thermo Fisher Scientific). A cell-free MTT blank was included on every plate to control for spectral interference and was normalized to DMSO to compute “Fa = 1 – (treated/vehicle)”. The median-effect parameters (m, Dm) were estimated by log-linear regression of log {Fa/(1-Fa)} vs. log (dose). At each combination point, Loewe dose equivalents (DxA, DxB) were derived from single-agent fits, and the combination index (CI) was calculated as CI = a/DxA + b/DxB calculated [13]. Isobolograms at Fa = 0.8 and 0.9 were obtained by linear interpolation along fixed-ratio series; doses were normalized by single-agent ED80 (u = Citarinostat/ED80_Citarinostat, v = VP/ED80_VP), with u + v = 1 as the additivity line [14]. For HAC2, we combined citarinostat with CA-3, a YAP–TEAD inhibitor [15], to rule out VP-specific off-target/artifact effects.
Immunofluorescence and image analysis
The cells were fixed 24 h after treatment, immunostained for YAP with DAPI nuclear counterstain, and imaged under identical exposure/gain and acquisition parameters across conditions. For each slide, five nonoverlapping fields were acquired. Image analysis was performed in Keyence Hybrid Cell Count using a single predefined template (fixed thresholds and size filters) applied uniformly to all images. Nuclei were segmented from the DAPI mask; nuclear YAP (n-YAP) was defined as the YAP signal within the nuclear mask for each cell. Total YAP (t-YAP) was quantified on the whole-cell mask, and cytoplasmic YAP (c-YAP) was calculated as t-YAP - n-YAP. The N/C ratio was computed per cell as the n-YAP/c-YAP ratio. To reduce optical bias, images were checked for saturation and subjected to uniform background subtraction. The t-YAP and N/C values were log-transformed, normalized to those of the DMSO control, and compared via two-tailed Welch’s t-tests.
Reverse transcription quantitative PCR (RT-qPCR)
Total RNA was extracted at 24 h after treatment, and reverse transcribed to cDNA. Quantitative PCR was performed for YAP, CTGF and CYR61 with KPNA6 as the reference gene, using a SYBR-Green chemistry on a real-time PCR system (LightCycler96, Roche). Cq values were analyzed by the ΔΔCt method with the DMSO control as the calibrator, and the results are shown as log2 fold changes [16].
Western blotting
Cells were lysed in ice-cold RIPA buffer (Wako) supplemented with a protease inhibitor cocktail (Wako). Lysates were incubated on ice for 30 min with brief vortexing every 5 min, followed by centrifugation at 14,000 × g for 20 min at 4 °C. Protein concentrations were determined using a BCA protein assay kit (Wako).
Equal amounts of protein (20 µg) were mixed with 4× sample buffer (Wako), heated at 95 °C for 3 min, and resolved on 10–12% SDS–polyacrylamide gels at 20 mA for 90 min. Proteins were transferred onto PVDF membranes (GE Healthcare Life Sciences) using semi-dry transfer at 140 mA for 1 h.
Membranes were blocked with Block ACE (WAKEN) for 1 h at room temperature and incubated overnight at 4 °C with a primary antibody against CTGF (rabbit polyclonal, 1:2000; Proteintech, #25474-1-AP) diluted in 10% Block ACE/TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20). After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:2000; Bio-Rad) for 1 h at room temperature and developed using enhanced chemiluminescence (ECL; Bio-Rad). Images were acquired with an ImageQuant 800 system (Cytiva), and band intensities were quantified using ImageQuant 800 TL software.
After CTGF detection, membranes were washed and reprobed with an anti–β-actin antibody (mouse monoclonal, 1:2000; MBL, #M177-3) diluted in TBS-T. β-actin was quantified using the same procedure. CTGF intensity was normalized to β-actin and expressed relative to control conditions.
Results
Clinical associations between HDAC6, YAP and CTGF expression in 171 OCCC tissues
In the tissue microarray, which included 171 cases with OCCC (Fig. 1, Figure S1), cytoplasmic YAP (c-YAP) was positive in 115 cases (67%), nuclear YAP (n-YAP) was positive in 127 cases (74%), cytoplasmic HDAC6 (c-HDAC6) was positive in 78 cases (46%), nuclear HDAC6 (n-HDAC6) was positive in 74 cases (43%), and CTGF was positive in 135 cases (79%).
Spearman’s correlation analysis revealed significant positive correlations between c-YAP and c-HDAC6, and between c-HDAC6 and CTGF, whereas c-HDAC6 and n-HDAC6 were significantly negatively correlated (Table 1).
Table 1.
Spearman’s rank correlation coefficient
Significant positive correlations were observed between c-YAP and c-HDAC6 and between c-HDAC6 and CTGF, whereas a significant negative correlation was observed between c-HDAC6 and n-HDAC6. No significant correlations were found among the other pairs
No other significant associations were observed among the tested pairs. Kaplan-Meier analysis (Figure S2) revealed no significant differences in OS or PFS between the expression groups (log-rank test, all p > 0.05). Consistently, univariate Cox regression analysis did not reveal any significant prognostic impact of these markers on OS or PFS (Table 2).
Table 2.
Univariate Cox proportional hazard models for overall survival and progression-free survival
(A) Overall survival, (B) Progression-free survival
None of the molecular markers were significantly associated with prognosis (all p > 0.05)
Since no variable reached significance in the univariate setting, multivariate Cox regression was not performed.
The combination of citarinostat and VP shows strong synergy across OCCC models
Across HAC2, OVTOKO, and RMG-1, fixed-ratio citarinostat and VP combinations yielded strong, high-Fa-biased synergy. Isobolograms indicated that dots were under the additivity line (isobole) for Fa = 0.8 and 0.9 in all lines (Fig. 2).
Fig. 2.
Normalized isobolograms at Fa = 0.8 and 0.9 showing significant synergy in all lines. Points denote mean interpolated dose pairs at Fa = 0.8 or 0.9. The coordinates are normalized such that u = Citarinostat/EDx_Citarinostat and v = VP/EDx_VP. The line u + v = 1 indicates Loewe additivity; u + v < 1 indicates synergy. (A–C) ED80: (A) HAC2, (B) OVTOKO, (C) RMG-1. (D–F) ED90: (D) HAC2, (E) OVTOKO, (F) RMG-1
The minimum CIs near that range were 0.11 (HAC2), 0.28 (OVTOKO), and 0.08 (RMG-1) (Figure S3, A-C). In HAC2, the combination of citarinostat and CA-3 had the same synergy (Figure S4).
The combination reduces YAP nuclear localization and modulates total YAP
Immunofluorescence revealed that, compared with DMSO, the combination treatment reduced the N/C YAP ratio in HAC2 cells (Fig. 3).
Fig. 3.
YAP immunofluorescence and quantitative compartmental analysis of HAC2. (A–D) Representative images: (A) DMSO, (B) citarinostat, (C) verteporfin, (D) combination. YAP (magenta); nuclei (DAPI, blue). The acquisition parameters (exposure/gain) were identical across the conditions. Scale bars show 100 μm. (E–F) Quantification of total YAP (t-YAP; whole-cell signal) (E) and the nuclear-to-cytoplasmic ratio (N/C; per-cell n-YAP/c-YAP) (F). The values were log2-transformed and normalized to those of the DMSO control (Δlog2 vs. DMSO). Verteporfin significantly decreased t-YAP, whereas the combination increased t-YAP; citarinostat had no significant effect on t-YAP. The N/C ratio was significantly lowered in all the treatments. Data was presented mean ± SEM (n = 5). Downward triangles mark off-scale data points (number indicated). *p < 0.05, **p < 0.005, ***p < 0.0005 (Welch’s t test vs. DMSO with correction)
In HAC2, VP decreased both t-YAP (p = 0.002) and the N/C YAP ratio (p = 0.016), whereas citarinostat did not change t-YAP but decreased the N/C ratio (p < 0.0001). With the combination, t-YAP was greater (p = 0.039), yet the N/C ratio was significantly lower (p = 0.014) than that of the DMSO control. OVTOKO presented the same trend, although the difference did not reach statistical significance (Figure S5).
mRNA levels of YAP, CTGF and CYR61 exhibit the same trends across all cell line
At 24 h, RT–qPCR revealed distinct transcript responses across the three OCCC cell lines (Fig. 4, Figure S6).
Fig. 4.
YAP and YAP-target gene expression in HAC2 cells after treatment with citarinostat and verteporfin. (A) mRNA expression of YAP, CTGF, and CYR61 in HAC2 cells. Values were log₂-transformed and expressed as Δlog₂ relative to the DMSO control. Citarinostat markedly increased CTGF expression, whereas YAP and CYR61 showed no significant changes. Verteporfin (VP) and the combination treatment did not significantly alter the expression of any of the three genes. (B) Representatives immunoblot of CTGF and β-actin in HAC2 cells treated with citarinostat, VP, or the combination. (C) Quantification of CTGF protein levels normalized to β-actin and expressed relative to the DMSO control. The combination treatment significantly reduced CTGF protein expression, whereas citarinostat and VP alone showed no significant effect. Data are presented as mean ± SEM (n = 3). *p < 0.05; ns, not significant (Welch’s t test vs. DMSO with correction)
YAP mRNA showed no statistically significant changes in any cell line. Across all lines, VP tended to increase YAP transcripts, whereas citarinostat and the combination consistently trended downward, although these changes were not significant. For CTGF, citarinostat significantly increased transcription in HAC2 and OVTOKO, while RMG-1 exhibited a similar but non-significant upward trend. In contrast, the combination treatment shifted CTGF expression downward in all cell lines, irrespective of significance. VP produced cell line–dependent, non-uniform effects. For CYR61, citarinostat significantly upregulated transcription in OVTOKO, and induced similar but non-significant increases in HAC2 and RMG-1. Across all lines, CYR61 expression under the combination treatment consistently trended downward, without reaching significance. As with CTGF, VP showed variable, line-dependent effects.
CTGF protein expression in HAC2 cells
In HAC2 cells, quantitative analysis normalized to DMSO showed that citarinostat produced a modest upward shift in CTGF protein abundance, whereas verteporfin tended to reduce CTGF levels; however, neither change reached statistical significance. In contrast, the combination treatment consistently suppressed CTGF expression and was the only condition to achieve a significant reduction (Fig. 4).
Discussion
We first conducted a tissue-level analysis in OCCC, which provided clinically grounded signals for pathway involvement. In a 171-case tissue microarray, c-HDAC6 correlated positively with c-YAP and CTGF, whereas c-HDAC6 and n-HDAC6 were inversely associated, and none of the markers predicted overall or progression-free survival. These associations, while not prognostic, pointed to an HDAC6-YAP-CTGF axis that is active in human tumors and therefore actionable. Because of these clinical observations, we performed mechanism-oriented evaluation in OCCC cell models to test whether dual HDAC6 and YAP–TEAD inhibition would generate therapeutic synergy.
Across three OCCC cell lines, fixed-ratio combinations of the HDAC6 inhibitor citarinostat and the YAP-TEAD-disrupting agent VP produced robust high-effect synergy. Isobolograms fell below the additivity isobole at Fa = 0.8–0.9, the Chou-Talalay combination indices were strongly synergistic (minimum CI ≈ 0.11–0.28). The synergy in high-Fa bias indicates synergy at effect levels that are most relevant for therapeutic translation [13, 17].
Mechanistically, the two agents appear to converge on the YAP axis through distinct but complementary nodes. In HAC2 cells, immunofluorescence showed that the combination increased t-YAP but reduced the N/C ratio, indicating restricted nuclear availability despite increased abundance. This pattern is consistent with the finding that HDAC6 modulates Hippo pathway activity and promotes cytoplasmic retention of YAP [10, 11]. VP alone decreased t-YAP, which is consistent with previous findings [18], whereas the combination increased t-YAP, likely reflecting compensatory upregulation because TEAD inhibition is known to elevate YAP expression [19, 20]. Importantly, the decrease in the N/C ratio was uniform across fields, supporting that the quantitative analysis, not the visual nuclear brightness, underlies our interpretation. The synergy reproduced by citarinostat and CA-3 strengthens the inference that TEAD engagement is the relevant mechanism rather than a VP artifact.
Across all three OCCC cell lines, transcript analysis showed a consistent pattern in which citarinostat alone tended to increase the transcription of CTGF and CYR61, whereas the combination shifted both genes toward decreased expression, regardless of statistical significance. This convergence of transcriptional trends across genetically distinct OCCC backgrounds supports a functional link between HDAC6 activity and the YAP–TEAD axis [21]. Importantly, in HAC2 cells, CTGF protein abundance largely reflected the transcript-level changes. Citarinostat increased CTGF protein levels without reaching statistical significance, while the combination significantly reduced CTGF. These findings suggest that, at least under these conditions, CTGF is regulated in a transcription-coupled manner [22–24].
These results are also consistent with our immunofluorescence analysis, in which citarinostat alone partially restricted YAP nuclear access, yet insufficiently to disrupt downstream transcriptional output. The addition of VP, a YAP–TEAD inhibitor, likely blocks YAP nuclear availability and transcriptional activity, thereby collapsing the compensatory YAP-dependent program induced by HDAC6 inhibition.
These findings support a model in which HDAC6 inhibition primes OCCC cells for YAP dependency, and dual HDAC6–YAP targeting induces coordinated disruption of YAP localization and function, providing a mechanistic basis for the high-effect synergy observed in our combination assays.
Translationally, this clinic-to-bench framework suggests that tumors with high cytoplasmic HDAC6 and YAP/CTGF positivity may be enriched for susceptibility to dual HDAC6 and YAP–TEAD targeting. Because both agents have clinical value [25–27], these findings support further evaluation in organoids, in vivo OCCC models and early-phase studies.
This study has limitations. First, in vivo validation is lacking; future work will test efficacy in patient-derived xenograft or orthotopic models. Second, our design focused on the genetic heterogeneity of OCCC, particularly ARID1A status, rather than on pathway-negative cellular backgrounds. The addition of HDAC6- or YAP-independent negative control cell lines would further strengthen pathway specificity and help exclude potential off-target effects. Third, we did not delineate the cell death modality; rescue experiments with pan caspase inhibitors and ferrostatin-1, alongside time-resolved Annexin V/PI or caspase assays, will clarify pathway contributions.
In summary, clinical profiling of OCCC revealed an HDAC6-YAP-CTGF axis that we then functionally explored in vitro. Dual HDAC6 and YAP-TEAD targeting restricts the nuclear access of YAP while blocking TEAD engagement, resulting in strong synergy across OCCC models. Our clinic-to-bench findings highlight the citarinostat-VP combination as a promising YAP-axis-directed therapeutic strategy in OCCC.
Supplementary Information
Below is the link to the electronic supplementary material.
Author contributions
SZ: conceptualization, data curation, formal analysis, investigation, methodology, writing – original draft. MiY: conceptualization, funding acquisition, investigation, resources, writing – review and editing. SS: data curation, investigation. MM: resources. MaY: investigation, resources, formal analysis, writing – review and editing. EK: formal analysis, writing - review and editing. KM: formal analysis, investigation, methodology, project administration, resources, supervision, writing – review and editing.
Funding
This study was supported by a grant from the Kanzawa Medical Research Foundation 2022.
Data availability
No datasets were generated or analyzed during the current study.
Declarations
Ethics approval
This study used the same OCCC tissue microarray cohort as our prior report under IRB approval No. 16–257 (Saitama Medical University International Medical Centre), in accordance with the Declaration of Helsinki. All study participants provided informed consent.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Supplementary Materials
Data Availability Statement
No datasets were generated or analyzed during the current study.