Human ovarian cancer cell lines SK-OV-3 and A2780 and 293T cells were obtained from the American Type Culture Collection (ATCC), and were maintained in high glucose DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 1% (v/v) penicillin/streptomycin (Beyotime Institute of Biotechnology) as previously described (17-19). All the cells were cultured at 37°C in a humidified chamber with 5% CO₂.

The SK-OV-3 derived OCSCs were generated as previously described (20). Briefly, SK-OV-3 cells were harvested and washed with FBS-free DMEM medium twice, then re-suspended and maintained under stem cell conditions by serum-free in DMEM medium supplemented with 5 mg/ml insulin (Sigma-Aldrich; Merck KGaA), 10 ng/ml human recombinant epidermal growth factor (Invitrogen; Thermo Fisher Scientific, Inc.), 10 ng/ml basic fibroblast growth factor (Invitrogen; Thermo Fisher Scientific, Inc.) and 0.3% bovine serum albumin (Sigma-Aldrich; Merck KGaA). Culture media were changed every 2 days by centrifuging the cells at 60 × g for 5 min at room temperature to remove the dead cell debris and SK-OV-3 cells proliferated as non-adherent spheres consequently in this condition. The cells of non-adherent spheres were subjected to identification of stem cell-like properties before following experiments.

Antibodies for ARID1A (mouse; cat. no. sc-32761), YAP (cat. no. sc-101199), CTGF (cat. no. sc-373936), CYR61 (cat. no. sc-374129), Nanog (cat. no. sc-293121), Sox2 (cat. no. sc-365823), Oct3/4 (cat. no. sc-5279), GAPDH (cat. no. sc-32233) and normal mouse IgG (cat. no. sc-2025) were purchased from Santa Cruz Biotechnology, Inc. Antibodies for p-YAP antibody (cat. no. AF5965), α-Tubulin (cat. no. AF0001), N-cadherin (cat. no. AF5237) and E-cadherin (cat. no. AF0138) were obtained from Beyotime Institute of Biotechnology. p-MST1 (cat. no. bs-3294R), MST1 (cat. no. bs-3504R), p-LATS1 (cat. no. bs-3245R), LATS1 (cat. no. bs-2904R), TAZ (cat. no. bs-12367R) and Vimentin (cat. no. bs-23063R) antibodies were purchased from BIOSS. Normal rabbit IgG (cat. no. 2729) and p-TAZ antibody (cat. no. 59971) were obtained from Cell Signaling Technology, Inc.

Plasmids expressing ARID1A-shRNA were generated by insertion with the hairpin shRNA templates of complementary oligonucleotides at the sites of XbaI and NotI into the shRNA expression vector, Pll3.7. ShRNA sequences used are as follows: shRNA sequence targeting ARID1A: 5'-AAC CAA AGT TAC TGT TGT TTA-3'; and a scrambled shRNA sequence (5'-TTT GTA CTA CAC AAA AGT ACTG-3') was used as control. Sequences were obtained from Shanghai Sangon Biotechnology Co. Ltd. Lentiviruses expressing ARID1A-shRNA or scrambled-shRNA were generated by co-transfecting the HEK293T packaging cells with lentiviral shRNA expression vector, and lentiviruses-containing supernatants with the titers greater than 1×10⁶ cfu/ml was used for infection of cells in the presence of 8 μg/ml polybrene (Sigma-Aldrich; Merck KGaA) as previously described (21).

Transient transfection was performed using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) with Opti-MEM (cat. no, 31985-062; Invitrogen; Thermo Fisher Scientific, Inc.) medium according to the manufacturer's instructions. Briefly, SK-OV-3 or A2780 cells were transfected with 10 μg indicated plasmids. At 12 h after transfection, the cells were cultured in fresh medium, and the cells were collected 48 h after transfection. Then the cellular lysates were subjected to reverse transcription-quantitative PCR (RT-qPCR), western blotting (WB) or other assays.

Total RNA was extracted from SK-OV-3 cells and A2780 cells by using a TRIzol reagent (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. 5 μg of total RNA was reversely transcribed by using SuperScript III reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and the oligo-(deoxythymidine) primer with incubation at 42°C for 1 h. After the termination of cDNA synthesis, mRNA levels of target genes were determined by RT-qPCR as previously described (21). Briefly, the initial denaturation was 95°C for 5 min; followed by 40 cycles of denaturation (95°C, 10 sec), annealing and extension (60°C, 30 sec). All reactions were conducted in a 20 μl reaction volume in triplicate. The relative expression of the mRNA levels was normalized to the GAPDH levels, and the relative difference in mRNA levels was calculated by the 2^-ΔΔCq method (22). The primers used were as follows: ARID1A forward, 5'-TCA TGC CCA ACC TTC GTA TC-3' and reverse, 5'-GAT GGC TGC TGG GAG TATG-3'; CTGF forward, 5'-CCT GTG CAG CAT GGA CGTT-3' and reverse, 5'-GGA CCA GGC AGT TGG CTC TAA-3'; CYR61 forward, 5'-CTC CCT GTT TTT GGA ATG GA-3' and reverse, 5'-TGG TCT TGC TGC ATT TCT TG-3'; Nanog forward, 5'-CCA TCC TTG CAA ATG TCT TCTG-3' and reverse, 5'-CTT TGG GAC TGG TGG AAG AATC-3'; Sox2 forward, 5'-GTG GTT ACC TCT TCC TCC CACT-3' and reverse, 5'-AGT GCT GGG ACA TGT GAA GTCT-3'; Oct3/4 forward, 5'-GTG GAG GAA GCT GAC AAC AATG-3' and reverse, 5'-AAT TCT CCA GGT TGC CTC TCA CT-3'; E-Cadherin forward, 5'-ACC AAC GAT AAT CCT CCG AT-3' and reverse, 5'-TCA GTG TGG TGA TTA CGA CG-3'; N-Cadherin forward, 5'-AAT CCT CCA GAG TTT ACT GC-3' and reverse, 5'-TCC TTA TCG GTC ACA GTT AG-3'; Vimentin forward, 5'-GAG AGG AAG CCG AAA ACAC-3' and reverse, 5'-TGC GTT CAA GGT CAA GACG-3'; and GAPDH forward, 5'-CCT GTT CGA CAG TCA GCCG-3' and reverse, 5'-CGA CCA AAT CCG TTG ACT CC-3'.

WB was performed using standard protocols as previously described (23). Briefly, cells were harvested and rinsed with pre-chilled PBS on ice, and then cell lysates were prepared using RIPA buffer (Beyotime Institute of Biotechnology). BCA protein assay kit was conducted to detect the concentration of protein. Generally, 50 μg of total protein was subjected to 8-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membrane (MilliporeSigma). Membranes were then blocked with 5% non-fat milk at room temperature for 1 h followed by incubation with different primary antibodies (1:1,000) at 4°C overnight. The membranes were then incubated with corresponding HRP-labeled Goat Anti-Rabbit IgG (H + L) (1:1,000 cat. no. A0208΄ Beyotime Institute of Biotechnology) for 1 h at room temperature the following day. Signals were subsequently detected using an ECL Kit (cat. no. P0018AS; Beyotime Institute of Biotechnology) according to the manufacturer's protocol.

SK-OV-3 or A2780 cells were washed with PBS and lysed with ice-cold extraction buffer (1% Triton-X 100, 10 mM EDTA, 1 mM PMSF, 1% Cocktail, pH=7.4). The soluble fraction was obtained by centrifuging cell lysates at 13,523 x g for 10 min at 4°C. The prepared supernatants were incubated at 4°C overnight with an ARID1A antibody, a TAZ antibody or control IgG. Then the mixture was incubated with 50 μl protein-A-agarose beads (Beyotime Institute of Biotechnology) for 2 h at 4°C, followed by three times of washing using lysis buffer. Immunoprecipitants were eluted with SDS loading buffer and resolved in SDS-PAGE gels. The proteins were transferred onto PVDF membranes and were further probed with appropriate antibodies correspondingly, referring to the a forementioned method in WB.

CCK-8 assay was performed as per the manufacturer's instructions (Shanghai Yeasen Biotechnology Co., Ltd.) as previously described (24). Briefly, 24 h after transfection, cells were seeded into 96-well plates at a cell density of ~4×10³ cells/well. At the selected time course after attachment, cells were incubated with 10 μl CCK-8 reagent for 2 h in the dark at 37°C, and optical density was consequently measured at a wavelength of 450 nm using a microplate reader (Tecan Group, Ltd.).

Wound healing assay was performed as previously described (15). Briefly, SK-OV-3 cells were seeded in six-well plate at 2×10⁵ cells/well and cultured for 24 h to confluence. Subsequently, cells were incubated with serum-free medium for 12 h. A sterile tip was used to scratch a straight line in each well. Then the wound gaps were monitored by light microscopy after 24 h, and were calculated using ImageJ software (National Institutes of Health). Experiments were performed for three independent experiments.

The colony formation assay was performed as previously described (23). A cell colony formed by the offspring of a single cell for >6 consecutive generations in vitro is called a colony, containing >50 cells. Briefly, SK-OV-3 cells were placed into a six-well plate at a density of ~500 cells/well, and were cultured at 37°C for two weeks. Colonies were then fixed with 4% paraformaldehyde (PFA) for 15 min and stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) in 2% ethanol for 10 min. After rinsing and drying, colonies were counted manually and images were captured. Each experiment was performed in triplicate.

Animal experiments were approved (approval no. 21045) by the Animal Ethics Committee of Zhejiang University (Zhejiang, China) and performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85-23, revised 1996). A total of 10 BALB/c nude mice (age, 4-5 weeks; weight, 18-20 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. and were bred under pathogen-free conditions (temperature, 18-22°C; humidity, 50-60%; 12/12-h light/dark cycle). The mice bedding, feed and water were replaced every 2 days. Mice were allocated into two groups: Control group and ARID1A group (n=5 in each group). Stably ARID1A-expressing viruses-infected (ARID1A) or control viruses-infected (Control) SK-OV-3 cells (1×10⁶ cells in 100 μl PBS) were injected into the right flank of mice (5×10⁶ cells per mouse) via subcutaneous injection. The volumes (V) of the xenograft tumors were examined every 3 days as follows: V = 0.5 x a x b², where 'a' indicates the long axis and 'b' indicates the short axis. After 21 days, the mice began to succumb and were immobile and rigid, and were not in a favorable mental state, the body weight was very low, and certain tumors reached the 1-1.5 cm in diameter. To reduce suffering, the mice were euthanized via intraperitoneal injection of 120 mg/kg pentobarbital sodium. Verification of death included cardiac and respiratory arrest, lack of reflexes and changes in mucosal color. The subcutaneous tumor tissues were consequently dissected and collected, and were weighed and used for subsequent examinations.

The collected xenograft tumor tissues were fixed in 4% PFA, embedded in paraffin, cut into sections with the thickness of 4 μm, dewaxed with xylene (Sinopharm Chemical Reagent Co., Ltd.) and rehydrated (100% ethanol for 3 min, 95% ethanol for 2 min, 80% ethanol for 2 min, 75% ethanol for 2 min, H₂O for 1 min). Then, the sections were treated with 0.01 M citrate buffer for antigen retrieval, incubated with 3% H₂O₂ solution at room temperature for 10 min, and incubated with 5% goat serum (Beyotime Institute of Biotechnology) at 37°C for 30 min. Next, the sections were incubated with the primary antibody against Ki67 (1:100; cat. no. ER1706-46; HUABIO) at 4°C overnight, and then with goat anti-rabbit IgG H&L (HRP) secondary antibody diluted at 1:1,000 at room temperature for 30 min next day. After DAB and hematoxylin staining, the sections were sealed with neutral resin and observed under a light microscope.

The results are presented as the mean ± SD. The SPSS 19.0 software program (IBM Corp.) and Excel (Excel 2016; Microsoft Corporation) were used for statistical analysis. Statistical significance of the data was analyzed by unpaired Student's t-test between two groups or with one-way ANOVA among multiple groups, followed by Dunnett's post hoc test. All P-values were two-sided, and P<0.05 was considered to indicate a statistically significant difference. All the experiments were repeated for a minimum of three times independently.

To explore the role of ARID1A in ovarian cancer cells, the ovarian cancer cell line SK-OV-3 was transfected with an ARID1A-expressing vector to ectopically express ARID1A and a vector expressing ARID1A-shRNA to suppress endogenous ARID1A expression. The results revealed that ARID1A overexpression significantly inhibited SK-OV-3 cell viability (Fig. 1A), whereas silencing ARID1A increased SK-OV-3 cell viability (Fig. 1B). Moreover, ARID1A overexpression suppressed SK-OV-3 cell migration and decreased colony numbers (Fig. 1C, D, G and H), whereas ARID1A knockdown significantly enhanced the migratory and colony formation abilities of SK-OV-3 cells (Fig. 1E, F, I and J). Consistent with these results, ARID1A-overexpression increased the expression of epithelial markers such as E-cadherin and decreased the expression of mesenchymal markers (including N-Cadherin and Vimentin), and stemness markers (such as Nanog, Oct3/4 and Sox2) (Fig. 1K and L). ARID1A knockdown resulted in the opposite effects (Fig. 1M and N), illustrating that ARID1A regulates EMT and stemness in ovarian cancer cells.

The Hippo pathway is closely associated with EMT and stemness in cancer cells (7,25). The potential effect of ARID1A on Hippo signaling was investigated to gain insight into the molecular mechanism connecting ARID1A expression with EMT and stemness in ovarian cancer cells. The protein levels and phosphorylation status of the main components of the Hippo pathway (MST1/2, LATS1/2, TAZ and YAP) (26) were analyzed in ovarian cancer cell lines expressing ARID1A or ARID1A-shRNA. The present results revealed that ARID1A overexpression resulted in a more active Hippo pathway in SK-OV-3 ovarian cancer cells, with an increased ratio of p-MST1/total MST1 and p-LATS1/total LATS1, compared with that in the control (Fig. 2A). Conversely, ARID1A-shRNA-expressing SK-OV-3 cells displayed a less active Hippo pathway with a decreased p-MST1/total MST1 to p-LATS1/total LATS1 ratio compared with the control cells (Fig. 2B). Consistent with this, p-TAZ and p-YAP levels were upregulated, but total TAZ and YAP expression was downregulated upon ARID1A-overexpression (Fig. 2C), whereas suppression of ARID1A expression by ARID1A-shRNA exerted the opposite effects (Fig. 2D). As expected, the mRNA and protein expression of TAZ/YAP-mediated target genes, including CTGF and CYR61, was significantly reduced by ARID1A overexpression and induced by ARID1A knockdown (Fig. 2E-H). This result was verified using the human ovarian cancer A2780 cell line, demonstrating that ARID1A affected CTGF and CYR61 mRNA expression (Fig. 2I and J). Thus, Hippo signaling activity was revealed to be regulated by ARID1A in ovarian cancer cells.

Cell density mediates Hippo pathway activity in cultured cells (27). To test the possible reciprocal effect of Hippo activity on ARID1A expression, SK-OV-3 cells were seeded at either low or high densities to deactivate or activate the Hippo pathway, respectively, which was estimated by examining the target gene CTGF and CYR61 expression (Fig. 3A). Activation of Hippo signaling by high cell density upregulated ARID1A protein expression (Fig. 3B), whereas ARID1A mRNA levels remained unchanged (Fig. 3B), suggesting that Hippo regulates ARID1A at a translational or post-translational level. As expected, activation of the Hippo pathway at high density weakened EMT and stemness in SK-OV-3 cells. This was associated with upregulated E-cadherin expression and downregulated expression of N-cadherin, Vimentin (Fig. 3C) and stemness markers (Nanog, Oct3/4 and Sox2) at both the mRNA and protein levels (Fig. 3D and E). By contrast, overexpression of TAZ, a key effector of the Hippo pathway, apparently reduced ARID1A protein expression (Fig. 3F) despite a slight alteration at the mRNA level (data not shown). At a culture condition with medium cell density, the TAZ-overexpressing ovarian cancer cells displayed upregulated EMT and stemness, including decreased E-cadherin expression and increased expression of N-Cadherin and Vimentin (Fig. 3F) and of stemness markers, including Nanog, Oct3/4 and Sox2 (Fig. 3G). Co-immunoprecipitation assays were performed to validate the possible regulation of ARID1A protein expression by Hippo. The present results showed an interaction between endogenous ARID1A and TAZ in SK-OV-3 cells, which could be verified using an ARID1A- or TAZ-antibody in A2780 cells (Fig. 3H-J), suggesting the potential mediation of ARID1A by TAZ. These results indicated that activation of Hippo signaling induces ARID1A expression and suppresses EMT and stemness via TAZ inhibition in ovarian cancer cells.

Given that ARID1A negatively regulates EMT and stemness in ovarian cancer cells but positively mediates the Hippo pathway, it was hypothesized that ARID1A affects EMT and stemness in ovarian cancer cells through the Hippo pathway. ARID1A overexpression significantly reduced TAZ-mediated viability, migration and colony formation in SK-OV-3 cells (Fig. 4A-E). Furthermore, overexpression of a gain-of-function mutant of TAZ (TAZ-S89) effectively countered the effects derived from ARID1A in ovarian cancer cells. This mutant has a mutation at Ser 89 (to Ala), rendering it resistant to phosphorylation by the upstream kinase LATS, and consequently, TAZ-S89 stably enters the nucleus and transactivates the downstream targets (28). The current data revealed that TAZ-S89 not only effectively reversed the ARID1A-suppressd cell viability (Fig. 4F) but mitigated the expression alterations of EMT and stemness markers induced by ARID1A overexpression, including the downregulation of E-cadherin and the upregulation of N-cadherin, Vimentin, Nanog, Oct3/4 and Sox2, compared with cells overexpressing ARID1A alone (Fig. 4G and H). Thus, activation of TAZ could attenuate the ARID1A-trigged suppression of EMT and stemness, reinforcing the notion that ARID1A negatively regulates EMT and stemness by activating the Hippo pathway.

SK-OV-3-derived OCSCs were isolated to further test the role of ARID1A in ovarian cancer stemness, as previously described (20). The isolated and cultured OCSCs displayed sphere-forming and stem cell-like phenotypes (Fig. 5A). The OCSCs possessed higher viability (Fig. 5B) and exhibited increased mRNA and protein expression of stemness markers Nanog, Oct3/4 and Sox2 (Fig. 5C and D), compared with the parent SK-OV-3 cells. By contrast, ARID1A expression was reduced in the OCSCs (Fig. 5E). However, overexpression of ARID1A in the OCSCs significantly downregulated the mRNA and protein expression of CTGF and CYR61 (Fig. 5F and G). It also lowered the EMT ability and stemness as determined by measuring the expression of E-cadherin, N-cadherin, Vimentin, and stemness markers Nanog, Oct3/4 and Sox2 (Fig. 5H and I). By contrast and as expected, ARID1A overexpression reduced OCSC migration and colony numbers (Fig. 5J-M).

Finally, ARID1A expression was determined by ARID1A-expressing lentiviruses in OCSCs (ARID1AOCSCs), which were subsequently injected into nude mice to form OCSCs-derived xenografts, to evaluate the roles of ARID1A in OCSCs' stemness in vivo. The volumes of control OCSCs and ARID1A-OCSC xenografts increased in a time-dependent manner within 21 days post-inoculation (Fig. 5N). However, no significant difference was observed between the body weights of mice bearing different xenografts (data not shown). Notably, the volumes of xenografts derived from control OCSCs were significantly higher than those of ARID1A-OCSCs from 15 to 21 days post-inoculation (Fig. 5N and O), and the xenograft-derived control OCSCs were heavier than the ARID1A-OCSCs group (Fig. 5P). Ki67 staining was then performed to investigate the proliferation of ARID1A-OCSCs xenografts. The present results demonstrated that ARID1A overexpression in OCSC-derived xenografts led to a significant decrease in the proliferation of tumor cells compared with that in the control OCSC-derived xenografts (Fig. 5Q). Thus, ARID1A was shown to inhibit stemness in OCSC xenografts.