Evaluating the specific STAT3 inhibitor YHO-1701 in ovarian cancer cell lines and patient-derived cell models: efficacy, mechanisms, and therapeutic potential

Sho Sato; Takahito Miura; Aiko Ogasawara; Daisuke Shintani; Shogo Yamaguchi; Hiroaki Inui; Akiko Yoshinaga; Masahiko Nishiyama; Momomi Tsugane; Kosei Hasegawa

doi:10.3802/jgo.2025.36.e24

. 2024 Aug 2;36(2):e24. doi: 10.3802/jgo.2025.36.e24

Evaluating the specific STAT3 inhibitor YHO-1701 in ovarian cancer cell lines and patient-derived cell models: efficacy, mechanisms, and therapeutic potential

Sho Sato ¹, Takahito Miura ², Aiko Ogasawara ¹, Daisuke Shintani ¹, Shogo Yamaguchi ¹, Hiroaki Inui ¹, Akiko Yoshinaga ², Masahiko Nishiyama ³, Momomi Tsugane ², Kosei Hasegawa ^1,^✉

PMCID: PMC11964963 PMID: 39129332

Abstract

Objective

Signal transducer and activator of transcription 3 (STAT3) plays key roles in regulating cancer cell proliferation, survival, and metastasis. We aimed to determine the effects of YHO-1701, an oral STAT3 inhibitor, in ovarian cancer (OC).

Methods

We evaluated the impact of YHO-1701 on cell growth in patient-derived cells (PDCs) and OC cell lines using standard cell proliferation assays. Spheroid models derived from PDCs were assessed using three-dimensional (3D) cell viability assays. Antitumor activity was performed in SKOV3 xenograft mice treated orally administrated YHO-1701 with 20 mg/kg. Changes in STAT3 signaling were analyzed by western blotting. The molecular mechanisms of STAT3 inhibition were investigated by sequencing RNA and analyzing pathways in the SKOV3 using a small interfering RNA targeting STAT3 (STAT3 siRNA) and YHO-1701.

Results

YHO-1701 inhibited the growth of OC cell lines by preventing STAT3 dimerization and decreasing the expression of its downstream signaling molecule, survivin. The growth of PDCs and spheroids obtained from patients with primary and recurrent OCs was significantly inhibited. Antitumor effect was observed in the SKOV3 xenograft mice with YHO-1701. YHO-1701 induced apoptosis in OC cells. Additionally, p53 and/or MAPK signaling pathways were upregulated in SKOV3 cells incubated with YHO-1701 and in those with STAT3 siRNA.

Conclusion

Our results showed that YHO-1701 suppressed cell growth in PDCs of OC, accompanied by survivin inhibition, and a decrease in the number of peritoneal metastasis in the mice by YHO-1701, compared with those treated with control. Therefore, YHO-1701 could be a promising candidate agent for treating OC.

Keywords: Epithelial Ovarian Cancer, Signal Transducer and Activator of Transcription 3

Synopsis

YHO-1701, an oral specific STAT3 inhibitor, inhibited the growth of patient-derived ovarian cancer cells. YHO-1701 inhibited the growth even in cells derived from patients with recurrence. YHO-1701 may induce p53-upregulation and/or MAPK-related signaling, suppressed survivin - the anti-apoptotic protein, and promoted apoptosis.

INTRODUCTION

Ovarian cancer (OC) is the leading cause of death among all gynecological cancers in adult women. According to the American Cancer Society, around 20,000 new patients are diagnosed with OC annually, resulting in over 12,000 deaths [1]. Over 60%–70% of OC patients who present with advanced or disseminated at the time of diagnosis, and they have a poor prognosis [2].

The standard treatment for OC involves a combination of cytoreductive surgery followed by platinum-based chemotherapy. Patients often initially respond well to primary chemotherapy. In patients with advanced OC, chemotherapy and PARP inhibitor maintenance (with/without bevacizumab) result in an increased progression-free survival, but not overall survival [3,4,5]. Advanced OC often recurs or multiple metastases develop over time. The most prevalent type of OC recurrence is peritoneal, which eventually leads to chemoresistance and death [6]. Thus, new strategies for treating OC with novel mechanisms of action are urgently needed.

Signal transducer and activator of transcription 3 (STAT3) plays crucial roles in regulating cell growth, survival, and metastasis, and constitutively functions in various types of solid cancers [7]. Phosphorylated STAT3 contributes to malignancy by upregulating the expression of pro-oncogenes such as survivin and promoting tumor cell survival [8,9,10]. Levels of phosphorylated (p) STAT3 are elevated in various malignancies. A systematic review and meta-analysis has revealed increased STAT3 or pSTAT3 expression in OC compared with healthy ovaries, as well as benign and borderline tumors [11].

Targeting STAT3 in OC has therapeutic potential. For instance, STAT3 activation is required for OC cell proliferation under hypoxia, and blocking STAT3 small interfering RNAs (STAT3 siRNAs) significantly reduces OC cell proliferation [12]. Knockdown of STAT3 also diminishes tumor proliferation and metastatic potential in mice injected with OC cells derived from ascites [13]. Thus, inhibiting STAT3 could be a promising strategy for managing OC.

Several STAT3 inhibitors have been evaluated in clinical trials [14,15], but they have not been approved for clinical use because of side and poor antitumor effects. Therefore, the development of new therapies targeting STAT3 is urgently needed. The first orally administered STAT3 dimerization inhibitor, STX-0119, directly bound to the SH2 region of the STAT3 monomer. This prevented dimerization and reduced the expression of downstream target genes involved in the STAT3 signaling pathway, including cyclin D1, c-myc, Bcl-xL, and survivin. Thus, STX-0119 was developed as a novel approach to STAT3 inhibition [16]. The novel quinoline carboxamide YHO-1701 derived from STX-0119, was then also developed as an oral STAT3 inhibitor [17]. The results of biochemical assays have shown that YHO-1701 has higher selectivity for blocking STAT3-SH2 binding to the phospho-Tyr peptide than STX-0119. Oral YHO-1701 exerts anti-tumor effects in mouse models bearing human oral squamous cell carcinoma cell (SAS) xenografts, with exposed to high concentrations of YHO-1701 for long periods [17].

The present study aimed to determine the actions of YHO-1701 in OC cell lines, patient-derived OC models and SKOV3 xenograft mice, and the underlying mechanisms through which YHO-1701 inhibits OC cell growth, to increase understanding of the potential role of the STAT3 pathway in OC.

MATERIALS AND METHODS

1. Cell lines and cell proliferation assays

We purchased OC cell lines from the respective suppliers as follows: A2780 (European Collection of Authenticated Cell Cultures [ECACC], Porton Down, UK), OVCAR3, SKOV3 and CAOV3 (American Type Culture Collection cell bank [ATCC], Manassas, VA, USA). The A2780, OVCAR3, and SKOV3 lines were cultured in RPMI 1640 (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest S.A.S., Nuaillé, France), and CAOV3 was cultured in Dulbecco’s Modified Eagle Medium (DMEM); Thermo Fisher Scientific Inc.) supplemented with 10% FBS. All cell lines were incubated at 37°C under a 5% CO₂ atmosphere.

OC cells (2.5×10³/well) were seeded in 96-well plates and incubated with 0.3, 1, 3, 10, 30 and 100 μM) YHO-1701 for 48 and 72 hours. Thereafter, cell proliferation was assessed using WST-8 assays (Kishida Chemical, Osaka, Japan) as described [18]. The 50% inhibitory concentration (IC₅₀) was calculated from dose-response curves.

2. Clinical tumor samples

All clinical tumor samples were obtained from patients with OC who were treated at Saitama Medical University International Medical Center between July 2017 and May 2020.

3. Isolation of patient-derived cells (PDCs) and cell proliferation assays

We isolated PDCs from surgical tissues and ascitic fluid samples obtained from patients with OC as described [19,20]. We seeded PDCs (5×10⁴/well) in 96-well collagen-coated plates and incubated them with 0, 0.3125, 1.25, 5, and 20 μM YHO-1701 for 72 hours. Cell proliferation was measured using CellTiter-Glo 2.0 Cell Viability Assays (Promega Corp., Madison, WI, USA).

4. Spheroid cultures and 3D cell proliferation assays

Fresh primary OC cells were incubated in ultra-low attachment culture dishes (Corning, NY, USA) as described [18]. Spheroid cells (2×10³/well) were seeded into ultra-low attachment 96-well plates and incubated with 0, 1, 10, and 100 μM YHO-1701 for 48 hours. Cell proliferation was evaluated using CellTiter-Glo® 3D Cell Viability Assay kits (Promega Corp.), as described [21].

5. Detection of dimer-form STAT3

Cells were treated with compounds for 24 hours and then lysed in an ice-cold isotonic buffer (20 mmol/L Tris [pH 7.0], 150 mmol/L NaCl, 6 mmol/L MgCl₂, 0.8 mmol/L PMSF and 20% glycerol). The lysates were separated on native-PAGE gels and immunoblotted with an anti-STAT3 antibody (Cell Signaling, Danvers, MA, USA) as described [17].

6. Western blotting

After incubating cells with YHO-1701 for 24 hours, proteins were extracted and western blotted as described [17]. Primary antibodies used in this analysis included anti-STAT3 and pSTAT3 Y705 (Cell Signaling, Danvers, MA, USA), survivin (R&D Systems Inc., Minneapolis, MN, USA) and anti-β-actin antibody (Sigma-Aldrich Corp., St. Louis, MO, USA).

7. Therapeutic evaluation of YHO-1701 in peritoneal dissemination mouse xenograft model

The antitumor efficacy of YHO-1701 was investigated in an SKOV3 xenograft model. The reasons of using SKOV3 for the peritoneal dissemination model are as follows. Several reports have shown that interleukin-6, which activates the STAT3 signal pathway, induces SKOV3 migration, invasion, and spheroids [22,23,24]. The SKOV3 xenograft model has also been used in another study on the antitumor effect of BBI608, another STAT3 inhibitor [25]. Female C.B-17/Icr-scid/scid Jcl (SCID) mice aged 6 weeks were purchased from CLEA Japan (Tokyo, Japan). An SKOV3 cell suspension was prepared at 1×10⁸ cells/mL using PBS, and 0.2 mL was injected intraperitoneally into the abdominal cavity of each SCID mouse. Based on the body weight three days following the tumor dissemination (day 1), the mice were randomly allocated to the following groups (n=6): a vehicle group and YHO-1701 monotherapy group (20 mg/kg). Treatment was started on day 1; the test compounds were administered orally using a 5-day-on/2-day-of×4 cycle schedule. Total peritoneal tumor weight and number of tumors were measured on Day 39. The antitumor efficacy was expressed based on tumor weight as of day 39 as the percentage tumor growth inhibition (% IR), calculated using the following formula:

IR (%)=(1−Mean Intraperitoneal Tumor Weight in the YHO-1701 Group/Mean Tumor Weight in the Control Group)×100

The body weight of each mouse was monitored twice a week to assess the tolerability of the YHO-1701 monotherapy. The relative body weight (RBW) on day n was calculated using the following formula:

RBW=Body Weight on Day n/Body Weight on Day 1

This animal study was conducted in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility in accordance with the Guidelines of the Yakult Central Institute and protocols approved by the Animal Experimental Committee of the Yakult Central Institute.

8. RNA interference assays

We purchased synthesized siRNA duplexes consisting of 19 nucleotides with 3′dTdT overhangs (Dharmacon, Lafayette, CO, USA, and QIAGEN, Venlo, Netherlands, Cat# SI02662338). The target sequence of the oligo STAT3 siRNAs was: 5′-CAGCCTCTCTGCAGAATTCAA-3′. We transfected SKOV3 cells, using with siRNA using Dharma FECT 1 siRNA transfection reagent (Dharmacon) as described by the manufacturer.

9. Apoptosis assays

Ovarian cell A2780, OVCAR3 and SKOV3 cells were incubated with 5 and 10 μM YHO-1701 for 24 hours, double stained using MEBCYTO^® Apoptosis Kits (Annexin V-FITC Kit) (MBL, Aichi, Japan) as described by the manufacturer, and analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, Inc., Brea, CA, USA).

10. RNA-sequencing

Total RNA (1 μg) was extracted from SKOV3 cells incubated with 5 μM YHO-1701 for 24 hours. We also transfected SKOV3 cells with STAT3 siRNA using NucleoSpin RNA (Takara-Bio, Shiga, Japan). Libraries were prepared from total RNA using TruSeq Stranded mRNA Sample Prep Kits (Illumina Inc., San Diego, CA, USA). Single-end 75 bp RNA sequencing proceeded using NextSeq500 High output v2 kits and a NextSeq500 (both from Illumina Inc.).

11. Pathway analysis

Low-quality reads were removed by quality control using fastp [26]. The remaining ≥96% reads were mapped to the human genome using STAR [27] (Ensembl database; reference genome: Homo_sapiens, GRCh38.dna.primary_assembly.fa.GZ). Transcript expression was quantified using RNA-Seq by Expectation-Maximization (RSEM) [28]. Genes with <100 reads in all samples were excluded from further analysis. Transcript expression was compared between control and samples incubated with YHO-1701, and between cells transfected with scramble siRNA and STAT3 siRNA using the R package DESeq2 [29]. Differentially expressed genes (DEGs) identified by YHO-1701 and siRNA analyses were categorized as upregulated or downregulated. Those with common fluctuations were extracted and mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using the R package fgsea [30]. Pathways with (false discovery rate)-corrected p<0.05 were considered significant and extracted.

12. Statistical analysis

Data were statistically analyzed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). Group comparisons were assessed by one-way ANOVA, Dunnett, and Student’s t-tests. All p-values were two-sided, and p<0.05 was considered statistically significant.

13. Ethical statement

This study proceeded according to the Declaration of Helsinki (2013 amendment) and was approved by the Institutional Review Board of Saitama Medical University International Medical Center (No. 12-096). Written and oral informed consent was obtained from each patient to participate in the study.

RESULTS

1. YHO-1701 inhibited growth of OC cell lines

We incubated the A2780, OVCAR3, SKOV3 and CAOV3 OC cell lines with 0.3, 1, 3, 10, 30 and 100 μM YHO-1701 for 48 and 72 hours to assess its impact on cell growth. Fig. 1A shows that YHO-1701 suppressed the growth of all four cell lines. The IC₅₀ of A2780, OVCAR3, SKOV3 and CAOV3 cells incubated with YHO-1701 for 48 hours (72 hours) was 0.90, 1.75, 14.84 and 5.83 (0.67, 0.98, 2.54 and 1.75 μM), respectively. We then investigated the impact of YHO-1701 on the STAT3 signaling pathways in these cell lines by western blotting. We found that YHO-1701 hindered STAT3 dimerization and downregulated pSTAT3 expression, which then dose-dependently reduced levels of downstream survivin in all four cell lines (Fig. 1B and C; Figs. S1, 2, 3, 4). These findings suggested that YHO-1701 inhibits OC cell growth by suppressing STAT3 signaling.

Fig. 1 — OC, ovarian cancer; STAT3, signal transducer and activator of transcription 3.

2. YHO-1701 inhibited the growth of PDCs

We evaluated the growth-inhibitory effect of YHO-1701 in a more clinically relevant context than established cell lines by isolating fresh PDCs from 34 patients with diverse histological types of OC who were admitted to our center. The PDCs were incubated without (vehicle) or with 0.31, 1.25, 5, and 20 μM YHO-1701 for 48 hours. Fig. 2A shows that all concentrations of YHO-1701 induced a significant growth inhibition (p<0.001). The median IC₅₀ was 2.9 (range: 0.5–9.0) μM. The ability of YHO-1701 to inhibit OC cell growth was notably independent of the original tumor location or Federation Internationale de Gynecologie et d'Obstetrique (FIGO) stage (Fig. 2B and C). However, resistance to YHO-1701 relatively greater by clear cell carcinoma than high-grade serous carcinoma (HGSC) (Fig. 2D). We investigated the ability of YHO-1701 to inhibit the growth of PDCs derived from the ascites of patients with recurrent OC. The median IC₅₀ was 2.9 (range: 1.1–5.0) μM. Table S1 summarizes the clinical characteristics and treatment history of the patients. Fig. 2E and F shows that YHO1701 notably inhibited the growth PDCs derived from patients who had undergone extensive chemotherapy. These findings emphasized the ability of YHO1701 to inhibit the growth of PDCs from patients with recurrent cancer as well as chemo-naïve patients undergoing primary surgery.

Fig. 2 — OC, ovarian cancer; PDC, patient-derived cell.

3. YHO-1701 inhibited the growth of patient-derived OC cells in spheroid models

Spheroid models provide a more physiologically relevant microenvironment that facilitates cell-cell and cell-matrix interactions, thus surpassing the limitations of traditional 2D cell culture. Hence, we established spheroid models using patient-derived OC cells and assessed the ability of YHO-1701 to inhibit anchorage-independent growth. Fig. 3 and Fig. S5 show the models developed from ascites of patients with platinum-refractory HGSC that developed during primary treatment (OC1), pre-treatment HGSC (OC2), and platinum-resistant, recurrent HGSC (OC3). Table S2 summarizes the clinical characteristics and treatment history of the patients. These PDCs formed spheroids under the culture conditions described in the Materials and Methods section. Incubation with YHO-1701 significantly inhibited the formation of all three PDCs. The IC₅₀ of OC1, OC2, and OC3 incubated with YHO-1701 for 72 hours was respectively, 14.3, 15.9 and 14.2 μM.

Fig. 3 — HE, hematoxylin and eosin; HGSC, high-grade serous carcinoma; IC₅₀, 50% inhibitory concentration; OC, ovarian cancer.

4. YHO-1701 suppressed intraperitoneal dissemination of SKOV3 cells in vivo

Here, we examined whether YHO-1701 could exhibit an antitumor effect in a mouse model of SKOV3 with peritoneal dissemination. In this model, tumor nodules derived from SKOV3 cells were mainly observed in the caudate lobe of the liver, left kidney, and pancreas, and sporadically observed in the diaphragm and right kidney on Day 39.

In accordance with the above-mentioned in vitro results, YHO-1701 induced tumor growth inhibition with an IR value of 32.2% (p=0.076) and led to a decrease in the number of tumor nodules (p<0.05) disseminated in the abdominal cavity (Fig. 4A and B). Notably, no apparent tumor was observed in one YHO-1701-treated mouse, which is suggestive of a complete response.

Fig. 4 — IR, inhibition rate; RBW, relative body weight.

We also monitored the systemic toxicity of YHO-1701 in the mice. There were no observed adverse effects. Moreover, our data showed that this therapy had little or no effect on the mice’s body weight throughout the experimental period (Fig. 4C and D).

5. YHO-1701 might induce upregulation of p53 and MAPK signaling pathway in SKOV3 cells

YHO-1701 exhibited an antitumor effect in SKOV3 xenograft mice. Furthermore, to gain insight in the molecular mechanism of STAT3 inhibition by YHO-1701 in the OC cell line, we analyzed the KEGG pathways of 2,102 DEGs in SKOV3 cells incubated with YHO-1701 and in those transfected with STAT3 siRNAs (Fig. 5A). The results revealed significantly upregulated p53 and MAPK signaling pathways in these cells under both conditions (Fig. 5B).

Fig. 5 — DEG, differentially expressed gene; DMSO, dimethyl sulfoxide (Control); KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; OC, ovarian cancer; siRNA, small interfering RNA.

6. Growth of OC cells was inhibited by YHO-1701 through promoting apoptosis

Downregulated survivin expression induced by YHO-1701 might contribute to apoptosis. p53- and/or MAPK-dependent signaling pathways upregulated by YHO-1701 may also be associated to apoptosis. We aimed to elucidate mechanisms underlying the growth inhibition of OC cells induced by YHO-1701 measuring apoptosis ratios in A2780, OVCAR3, and SKOV3 OC cells using flow cytometry. Fig. 6 shows that YHO-1701 dose-dependently increased the ratios (%) of apoptotic OC cells, indicating that it enhanced apoptosis. These findings suggested that the cell growth inhibition induced by YHO-1701 is at least partly mediated by the induction of apoptosis.

Fig. 6 — OC, ovarian cancer; PI, propidium iodide.

DISCUSSION

We aimed to determine the ability of the novel STAT3 inhibitor, YHO-1701, to inhibit the growth of OC cells, to decrease the tumor volume in mice with disseminated OC, and the underlying mechanisms to better understand the potential role of the STAT3 pathway in OC. Our findings revealed that YHO-1701 inhibited the growth of OC cell lines and PDCs, including spheroid formation, and those derived from patients who had undergone several rounds of intensive therapy. YHO-1701 also showed an antitumor effect in the dissemination mouse model of OC. We found that YHO-1701 suppressed survivin expression, which contributed to enhanced OC cell apoptosis. Additionally, we showed that p53- and/or MAPK-dependent signaling pathways upregulated by YHO-1701 may be associated to OC cell apoptosis.

Some findings have indicated that STAT3 inhibition has potential as a promising approach for suppressing OC [13,31]. Recent sequencing of RNA in single cells obtained from ascites samples of HGSC revealed the expression of JAK/STAT3 in malignant cells and cancer-associated fibroblasts, and that inhibiting this pathway resulted in potent anti-tumor activity in PDC cultures and xenograft models [32]. Additionally, a study of the effects of export protein 1 (XPO1) and survivin on OC cell proliferation and apoptosis has shown that reducing survivin expression alleviates OC progression [33]. Previous novel STAT3 inhibitors have suppressed tumor growth in preclinical models of OC [13,34]. Of particular relevance, YHO-1701 has shown significant anti-tumor activity by interfering with multistep events, including STAT3 dimerization, in the SAS cells [17]. To the best of our knowledge, this is the first study to determine the ability of YHO-1701 to inhibit OC cell growth using PDCs, spheroid models and SKOV3 xenograft models.

Spheroid models can reproduce the original tumor environment and its characteristics, including drug sensitivity [21]. Although YHO1701 significantly inhibited spheroid formation in all three PDCs, the IC₅₀ was higher than that of PDCs obtained from 2D cell cultures. This disparity could be attributed to differences in cell types or the inherent resistance of cells within the spheroid structure, which might require higher concentrations of cytotoxin than 2D cultures, because the structural integrity is preserved and the viability of cells in enhanced in spheroid models [32].

We confirmed the antitumor effect in OC with YHO-1701 not only in in vitro models, but also in in vivo models. We confirmed with increasing no specific toxicity for SKOV3 xenograft mice with YHO-1701 as previous in vivo studies with using cancer cell line xenograft mice, including human oral cancer cell line, melanoma cell line, cutaneous T-cell lymphoma cell line, and the ALK-rearranged lung cancer cell line [17,35].

Although Yaginuma and Westphal [36] reported that there was no expression of p53 protein due to the presence of sequence deletions / rearrangements in at least one allele of the p53 gene in SKOV3, genes associated with the p53 signaling pathway were significantly upregulated in SKOV3 incubated with both YHO-1701 and siSTAT3 in this study. Selvendiran et al. [12] also reported that HO-3867, STAT3 inhibitor, induced the expression of p53 and apoptosis in A2780. Our result may support them. And there is the report that the inhibition of STAT3 in cancer cells increases p53 expression and triggers tumor cell apoptosis through p53-mediated mechanisms [37]. Here, we confirmed that YHO-1701 suppressed survivin expression and induced apoptosis in OC cells. An association between wild-type p53 expression and powerful inhibition of the survivin promoter has been identified in various cell types [38].

The inhibition of STAT3 also resulted in the upregulation of genes involved in the MAPK signaling pathway, which also may contribute to YHO-1701-induced apoptosis potentially. The MAPK pathway is crucial for intracellular signaling systems associated with growth, development, and apoptosis. Several studies have provided evidence of an association between STAT3 and MAPK signaling pathways. The expression of phosphorylated ERK (pERK) and STAT3 (pSTAT3) correlates inversely in pancreatic cancer cells [39], as does that of pERK1/2 and pSTAT3 in esophageal squamous cell carcinoma [40].

Our study has several limitations. First, although we had showed that upregulated p53- and/or MAPK-dependent signaling pathways in YHO-1701 treated OC cell and siSTAT3 OC cell, we had not performed the experiments to confirm that increase the expression of signaling proteins in these cells. Our findings that YHO-1701 induces upregulated p53- and/or MAPK-dependent signaling pathways indicated possibility, we need the further experiments to validate them. Next, we do not have the answer if YHO-1701 had antitumor effect either dependent on HRD or BRCA status, because the cases of this study were not performed HRD test and BRCA analysis.

In conclusion, our findings highlighted the ability of YHO-1701 to inhibit the growth of OC cells. We showed that YHO-1701 induces apoptosis in OC cells by downregulating anti-apoptotic survivin. These results pave the way for further preclinical and clinical evaluations of YHO-1701 as a candidate for treating OC.

ACKNOWLEDGEMENTS

We express our gratitude to Ms. Mayu Yasuno and Ms. Yuko Ijima for valuable support with sample collection and Ms. Chiharu Shimizu and Ms. Akiko Iwasa for exceptional technical assistance.

Footnotes

Conflict of Interest: No potential conflict of interest relevant to this article was reported.

This study was partly sponsored by Yakult Honsha Co. Ltd. (Tokyo, Japan)

Author Contributions:

Conceptualization: S.S., O.A., S.D., T.M., H.K.
Data curation: S.S., M.T., O.A., S.D., Y.S., I.H., Y.A., N.M., T.M., H.K.
Formal analysis: S.S., M.T., Y.S., I.H., Y.A., N.M., T.M., H.K.
Funding acquisition: S.S., H.K.
Investigation: S.S., S.D., Y.S., I.H., Y.A., N.M., T.M., H.K.
Methodology: S.S., Y.A., N.M., T.M.
Project administration: S.S., S.D., H.K.
Resources: S.S., M.T., O.A., H.K.
Supervision: S.S., M.T., H.K.
Validation: S.S., M.T., H.K.
Visualization: S.S., H.K.
Writing - original draft: S.S., H.K.
Writing - review & editing: S.S., H.K.

SUPPLEMENTARY MATERIALS

Table S1

Clinical characteristics and treatment history of patients with recurrent ovarian cancer

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Table S2

Clinical characteristics and treatment history of patients with established 3D culture

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Fig. S1

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on A2780. Data are presented as the mean ± standard deviation.

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Fig. S2

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on OVCAR3. Data are presented as the mean ± standard deviation.

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Fig. S3

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on CAOV3. Data are presented as the mean ± standard deviation.

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Fig. S4

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on SKOV3. Data are presented as the mean ± standard deviation.

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Fig. S5

Representative images of established patient-derived OC cell in spheroid model OC1 with control, 10 μM and 100 μM YHO-1701 for 24 hours and 48 hours.

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14.Bendell JC, Hong DS, Burris HA, 3rd, Naing A, Jones SF, Falchook G, et al. Phase 1, open-label, dose-escalation, and pharmacokinetic study of STAT3 inhibitor OPB-31121 in subjects with advanced solid tumors. Cancer Chemother Pharmacol. 2014;74:125–130. doi: 10.1007/s00280-014-2480-2. [DOI] [PubMed] [Google Scholar]
15.Ogura M, Uchida T, Terui Y, Hayakawa F, Kobayashi Y, Taniwaki M, et al. Phase I study of OPB-51602, an oral inhibitor of signal transducer and activator of transcription 3, in patients with relapsed/refractory hematological malignancies. Cancer Sci. 2015;106:896–901. doi: 10.1111/cas.12683. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Matsuno K, Masuda Y, Uehara Y, Sato H, Muroya A, Takahashi O, et al. Identification of a new series of STAT3 inhibitors by virtual screening. ACS Med Chem Lett. 2010;1:371–375. doi: 10.1021/ml1000273. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Nishisaka F, Taniguchi K, Tsugane M, Hirata G, Takagi A, Asakawa N, et al. Antitumor activity of a novel oral signal transducer and activator of transcription 3 inhibitor YHO-1701. Cancer Sci. 2020;111:1774–1784. doi: 10.1111/cas.14369. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Haibara H, Yamazaki R, Nishiyama Y, Ono M, Kobayashi T, Hokkyo-Itagaki A, et al. YPC-21661 and YPC-22026, novel small molecules, inhibit ZNF143 activity in vitro and in vivo. Cancer Sci. 2017;108:1042–1048. doi: 10.1111/cas.13199. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Ikeda Y, Park JH, Miyamoto T, Takamatsu N, Kato T, Iwasa A, et al. T-LAK cell-originated protein kinase (TOPK) as a prognostic factor and a potential therapeutic target in ovarian cancer. Clin Cancer Res. 2016;22:6110–6117. doi: 10.1158/1078-0432.CCR-16-0207. [DOI] [PubMed] [Google Scholar]
20.Ikeda Y, Sato S, Yabuno A, Shintani D, Ogasawara A, Miwa M, et al. High expression of maternal embryonic leucine-zipper kinase (MELK) impacts clinical outcomes in patients with ovarian cancer and its inhibition suppresses ovarian cancer cells growth ex vivo. J Gynecol Oncol. 2020;31:e93. doi: 10.3802/jgo.2020.31.e93. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Shiba S, Ikeda K, Suzuki T, Shintani D, Okamoto K, Horie-Inoue K, et al. Hormonal regulation of patient-derived endometrial cancer stem-like cells generated by three-dimensional culture. Endocrinology. 2019;160:1895–1906. doi: 10.1210/en.2019-00362. [DOI] [PubMed] [Google Scholar]
22.So KA, Min KJ, Hong JH, Lee JK. Interleukin-6 expression by interactions between gynecologic cancer cells and human mesenchymal stem cells promotes epithelial-mesenchymal transition. Int J Oncol. 2015;47:1451–1459. doi: 10.3892/ijo.2015.3122. [DOI] [PubMed] [Google Scholar]
23.Ding DC, Liu HW, Chu TY. Interleukin-6 from ovarian mesenchymal stem cells promotes proliferation, sphere and colony formation and tumorigenesis of an ovarian cancer cell line SKOV3. J Cancer. 2016;7:1815–1823. doi: 10.7150/jca.16116. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Kim S, Gwak H, Kim HS, Kim B, Dhanasekaran DN, Song YS. Malignant ascites enhances migratory and invasive properties of ovarian cancer cells with membrane bound IL-6R in vitro. Oncotarget. 2016;7:83148–83159. doi: 10.18632/oncotarget.13074. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Li H, Qian Y, Wang X, Pi R, Zhao X, Wei X. Targeted activation of Stat3 in combination with paclitaxel results in increased apoptosis in epithelial ovarian cancer cells and a reduced tumour burden. Cell Prolif. 2020;53:e12719. doi: 10.1111/cpr.12719. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–i890. doi: 10.1093/bioinformatics/bty560. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. doi: 10.1186/1471-2105-12-323. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Korotkevich G, Sukhov V, Budin N, Shpak B, Artyomov MN, Sergushichev A. Fast gene set enrichment analysis. bioRxiv. 2021 [Google Scholar]
31.Selvendiran K, Bratasz A, Kuppusamy ML, Tazi MF, Rivera BK, Kuppusamy P. Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3. Int J Cancer. 2009;125:2198–2204. doi: 10.1002/ijc.24601. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Izar B, Tirosh I, Stover EH, Wakiro I, Cuoco MS, Alter I, et al. A single-cell landscape of high-grade serous ovarian cancer. Nat Med. 2020;26:1271–1279. doi: 10.1038/s41591-020-0926-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Zhang J, Xu X, Chen Y, Guan X, Zhu H, Qi Y. The abnormal expression of chromosomal region maintenance 1 (CRM1)-survivin axis in ovarian cancer and its related mechanisms regulating proliferation and apoptosis of ovarian cancer cells. Bioengineered. 2022;13:624–633. doi: 10.1080/21655979.2021.2012416. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
34.Wen W, Liang W, Wu J, Kowolik CM, Buettner R, Scuto A, et al. Targeting JAK1/STAT3 signaling suppresses tumor progression and metastasis in a peritoneal model of human ovarian cancer. Mol Cancer Ther. 2014;13:3037–3048. doi: 10.1158/1535-7163.MCT-14-0077. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Yanagimura N, Takeuchi S, Fukuda K, Arai S, Tanimoto A, Nishiyama A, et al. STAT3 inhibition suppresses adaptive survival of ALK-rearranged lung cancer cells through transcriptional modulation of apoptosis. NPJ Precis Oncol. 2022;6:11. doi: 10.1038/s41698-022-00254-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Yaginuma Y, Westphal H. Abnormal structure and expression of the p53 gene in human ovarian carcinoma cell lines. Cancer Res. 1992;52:4196–4199. [PubMed] [Google Scholar]
37.Niu G, Wright KL, Ma Y, Wright GM, Huang M, Irby R, et al. Role of Stat3 in regulating p53 expression and function. Mol Cell Biol. 2005;25:7432–7440. doi: 10.1128/MCB.25.17.7432-7440.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S, et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene. 2002;21:2613–2622. doi: 10.1038/sj.onc.1205353. [DOI] [PubMed] [Google Scholar]
39.Nagathihalli NS, Castellanos JA, Lamichhane P, Messaggio F, Shi C, Dai X, et al. Inverse correlation of STAT3 and MEK signaling mediates resistance to RAS pathway inhibition in pancreatic cancer. Cancer Res. 2018;78:6235–6246. doi: 10.1158/0008-5472.CAN-18-0634. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Zheng ZY, Chu MY, Lin W, Zheng YQ, Xu XE, Chen Y, et al. Blocking STAT3 signaling augments MEK/ERK inhibitor efficacy in esophageal squamous cell carcinoma. Cell Death Dis. 2022;13:496. doi: 10.1038/s41419-022-04941-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1

Clinical characteristics and treatment history of patients with recurrent ovarian cancer

jgo-36-e24-s001.xls^{(28KB, xls)}

Table S2

Clinical characteristics and treatment history of patients with established 3D culture

jgo-36-e24-s002.xls^{(27KB, xls)}

Fig. S1

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on A2780. Data are presented as the mean ± standard deviation.

jgo-36-e24-s003.ppt^{(562KB, ppt)}

Fig. S2

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on OVCAR3. Data are presented as the mean ± standard deviation.

jgo-36-e24-s004.ppt^{(554KB, ppt)}

Fig. S3

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on CAOV3. Data are presented as the mean ± standard deviation.

jgo-36-e24-s005.ppt^{(560KB, ppt)}

Fig. S4

Statistical analysis of the protein expression levels of pSTAT3, STAT3, survivin and beta-actin relative to control on SKOV3. Data are presented as the mean ± standard deviation.

jgo-36-e24-s006.ppt^{(549.5KB, ppt)}

Fig. S5

Representative images of established patient-derived OC cell in spheroid model OC1 with control, 10 μM and 100 μM YHO-1701 for 24 hours and 48 hours.

jgo-36-e24-s007.ppt^{(1.2MB, ppt)}

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[B18] 18.Haibara H, Yamazaki R, Nishiyama Y, Ono M, Kobayashi T, Hokkyo-Itagaki A, et al. YPC-21661 and YPC-22026, novel small molecules, inhibit ZNF143 activity in vitro and in vivo. Cancer Sci. 2017;108:1042–1048. doi: 10.1111/cas.13199. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19.Ikeda Y, Park JH, Miyamoto T, Takamatsu N, Kato T, Iwasa A, et al. T-LAK cell-originated protein kinase (TOPK) as a prognostic factor and a potential therapeutic target in ovarian cancer. Clin Cancer Res. 2016;22:6110–6117. doi: 10.1158/1078-0432.CCR-16-0207. [DOI] [PubMed] [Google Scholar]

[B20] 20.Ikeda Y, Sato S, Yabuno A, Shintani D, Ogasawara A, Miwa M, et al. High expression of maternal embryonic leucine-zipper kinase (MELK) impacts clinical outcomes in patients with ovarian cancer and its inhibition suppresses ovarian cancer cells growth ex vivo. J Gynecol Oncol. 2020;31:e93. doi: 10.3802/jgo.2020.31.e93. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Shiba S, Ikeda K, Suzuki T, Shintani D, Okamoto K, Horie-Inoue K, et al. Hormonal regulation of patient-derived endometrial cancer stem-like cells generated by three-dimensional culture. Endocrinology. 2019;160:1895–1906. doi: 10.1210/en.2019-00362. [DOI] [PubMed] [Google Scholar]

[B22] 22.So KA, Min KJ, Hong JH, Lee JK. Interleukin-6 expression by interactions between gynecologic cancer cells and human mesenchymal stem cells promotes epithelial-mesenchymal transition. Int J Oncol. 2015;47:1451–1459. doi: 10.3892/ijo.2015.3122. [DOI] [PubMed] [Google Scholar]

[B23] 23.Ding DC, Liu HW, Chu TY. Interleukin-6 from ovarian mesenchymal stem cells promotes proliferation, sphere and colony formation and tumorigenesis of an ovarian cancer cell line SKOV3. J Cancer. 2016;7:1815–1823. doi: 10.7150/jca.16116. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Kim S, Gwak H, Kim HS, Kim B, Dhanasekaran DN, Song YS. Malignant ascites enhances migratory and invasive properties of ovarian cancer cells with membrane bound IL-6R in vitro. Oncotarget. 2016;7:83148–83159. doi: 10.18632/oncotarget.13074. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B26] 26.Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–i890. doi: 10.1093/bioinformatics/bty560. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28.Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. doi: 10.1186/1471-2105-12-323. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Korotkevich G, Sukhov V, Budin N, Shpak B, Artyomov MN, Sergushichev A. Fast gene set enrichment analysis. bioRxiv. 2021 [Google Scholar]

[B31] 31.Selvendiran K, Bratasz A, Kuppusamy ML, Tazi MF, Rivera BK, Kuppusamy P. Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3. Int J Cancer. 2009;125:2198–2204. doi: 10.1002/ijc.24601. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32.Izar B, Tirosh I, Stover EH, Wakiro I, Cuoco MS, Alter I, et al. A single-cell landscape of high-grade serous ovarian cancer. Nat Med. 2020;26:1271–1279. doi: 10.1038/s41591-020-0926-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33.Zhang J, Xu X, Chen Y, Guan X, Zhu H, Qi Y. The abnormal expression of chromosomal region maintenance 1 (CRM1)-survivin axis in ovarian cancer and its related mechanisms regulating proliferation and apoptosis of ovarian cancer cells. Bioengineered. 2022;13:624–633. doi: 10.1080/21655979.2021.2012416. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]

[B34] 34.Wen W, Liang W, Wu J, Kowolik CM, Buettner R, Scuto A, et al. Targeting JAK1/STAT3 signaling suppresses tumor progression and metastasis in a peritoneal model of human ovarian cancer. Mol Cancer Ther. 2014;13:3037–3048. doi: 10.1158/1535-7163.MCT-14-0077. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35.Yanagimura N, Takeuchi S, Fukuda K, Arai S, Tanimoto A, Nishiyama A, et al. STAT3 inhibition suppresses adaptive survival of ALK-rearranged lung cancer cells through transcriptional modulation of apoptosis. NPJ Precis Oncol. 2022;6:11. doi: 10.1038/s41698-022-00254-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Yaginuma Y, Westphal H. Abnormal structure and expression of the p53 gene in human ovarian carcinoma cell lines. Cancer Res. 1992;52:4196–4199. [PubMed] [Google Scholar]

[B37] 37.Niu G, Wright KL, Ma Y, Wright GM, Huang M, Irby R, et al. Role of Stat3 in regulating p53 expression and function. Mol Cell Biol. 2005;25:7432–7440. doi: 10.1128/MCB.25.17.7432-7440.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38.Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S, et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene. 2002;21:2613–2622. doi: 10.1038/sj.onc.1205353. [DOI] [PubMed] [Google Scholar]

[B39] 39.Nagathihalli NS, Castellanos JA, Lamichhane P, Messaggio F, Shi C, Dai X, et al. Inverse correlation of STAT3 and MEK signaling mediates resistance to RAS pathway inhibition in pancreatic cancer. Cancer Res. 2018;78:6235–6246. doi: 10.1158/0008-5472.CAN-18-0634. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40.Zheng ZY, Chu MY, Lin W, Zheng YQ, Xu XE, Chen Y, et al. Blocking STAT3 signaling augments MEK/ERK inhibitor efficacy in esophageal squamous cell carcinoma. Cell Death Dis. 2022;13:496. doi: 10.1038/s41419-022-04941-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Evaluating the specific STAT3 inhibitor YHO-1701 in ovarian cancer cell lines and patient-derived cell models: efficacy, mechanisms, and therapeutic potential

Sho Sato

Takahito Miura

Aiko Ogasawara

Daisuke Shintani

Shogo Yamaguchi

Hiroaki Inui

Akiko Yoshinaga

Masahiko Nishiyama

Momomi Tsugane

Kosei Hasegawa

Abstract

Objective

Methods

Results

Conclusion

Synopsis

INTRODUCTION

MATERIALS AND METHODS

1. Cell lines and cell proliferation assays

2. Clinical tumor samples

3. Isolation of patient-derived cells (PDCs) and cell proliferation assays

4. Spheroid cultures and 3D cell proliferation assays

5. Detection of dimer-form STAT3

6. Western blotting

7. Therapeutic evaluation of YHO-1701 in peritoneal dissemination mouse xenograft model

8. RNA interference assays

9. Apoptosis assays

10. RNA-sequencing

11. Pathway analysis

12. Statistical analysis

13. Ethical statement

RESULTS

1. YHO-1701 inhibited growth of OC cell lines

2. YHO-1701 inhibited the growth of PDCs

3. YHO-1701 inhibited the growth of patient-derived OC cells in spheroid models

4. YHO-1701 suppressed intraperitoneal dissemination of SKOV3 cells in vivo

5. YHO-1701 might induce upregulation of p53 and MAPK signaling pathway in SKOV3 cells

Fig. 5. Molecular mechanism of YHO-1701-mediated STAT3 inhibition in OC cell line. (A) Kyoto Encyclopedia of Genes and Genomes pathway analysis of 2,102 DEGs between SKOV3 cells incubated with YHO-1701 and those transfected with siRNAs targeting STAT3. (B) Results of KEGG pathway analysis.

6. Growth of OC cells was inhibited by YHO-1701 through promoting apoptosis

Fig. 6. Induction of OC cell apoptosis mediated by YHO-1701. (A) Apoptotic A2780, OVCAR3 and SKOV3 OC cells incubated with 5 and 10 μM YHO-1701 for 24 hours, respectively. (B) Apoptotic cell numbers were calculated based on flow cytometry results.

DISCUSSION

ACKNOWLEDGEMENTS

Footnotes

SUPPLEMENTARY MATERIALS

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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