A total of 149 ovarian cancer tissue samples (patient age range, 34–79 years; median age, 56 years) and 64 healthy fallopian tube epithelial tissues (patient age range, 26–74 years; median age, 47 years) with detailed clinical information were collected from the Pathology Department at Qilu Hospital of Shandong University (Ji'nan, China) between January 2005 and January 2015. All the malignant samples were diagnosed in accordance with the International Federation of Gynecology and Obstetrics criteria (36). The healthy samples were collected from patients who underwent surgery for benign conditions. Signed consents were collected from all the patients and the study was approved by the Ethics Committee of Qilu Hospital of Shandong University.

Survival analysis was performed on datasets from the Gene Expression Omnibus (GEO) database, including 523 patients for overall survival analysis using datasets GSE18520 (37), GSE26193 (38), GSE30161 (39), GSE63885 (40) and GSE9891 (41), and 483 patients for the progression-free survival analysis using datasets GSE26193, GSE30161, GSE63885, GSE9891, GSE65986 (42) on Kaplan-Meier Plotter (43).

Human ovarian cancer cells A2780 (cat. no. CL-0013; Procell Life Science & Technology Co., Ltd.) were cultured in RPMI-1640 medium supplemented with 10% FBS and penicillin (100 IU/ml) and streptomycin (100 µg/ml) (all Gibco; Thermo Fisher Scientific, Inc.). HEY cells (gifted from Dr Jianjun Wei; Laboratory at Northwestern University) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. All the cells were maintained in a humidified incubator at 37°C with 5% CO₂.

IHC staining of the tissue microarray (TMA) was performed on 4-µm sections sliced from each TMA receiver block fixed with 4% paraformaldehyde at room temperature for 48 h and embedded in paraffin. Tissue slides were deparaffinized in xylene and rehydrated in a graded series of ethanol (10 min each in 100, 95, 80 and 70% ethanol). Antigen retrieval was performed using a heat-induced epitope retrieval method with 10 mmol/l EDTA buffer (pH 8.0) at 98°C for 15 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol for 15 min at 37°C, and non-specific binding was blocked by incubation with donkey serum as part of the SP9000 IHC kit (OriGene Technologies, Inc.; cat. no. SP9000) for 30 min at 37°C. The slides were subsequently incubated overnight at 4°C in a humid chamber with anti-IQGAP3 (Abcam; cat. no. ab219354) antibody at a dilution of 3 µg/ml. Staining was visualized using I–View 3,3′-diaminobenzidine staining detection system (OriGene Technologies, Inc.; cat. no. ZLI-9018). The IHC score was determined using a semi-quantitative method based on the extent and intensity of positively stained cells. The percentage of positive cells within each sample was scored independently from 0 to 100% upon observation under a light microscope (magnification, ×10). The intensity of immunostaining was graded as follows: 0, Negative; 1, weak; 2, moderate; and 3, strong. The final IHC score was generated by multiplying the percentage extent with the staining intensity score. Then, two gynecological pathologists independently reviewed the IHC staining. High IQGAP3 expression grade was defined as a final IHC score ≥100.

For stable transfection, lentiviral vector GV493 (hU6-MCS-CBh-gcGFP-IRES-puromycin) was packaged with IQGAP3 short hairpin (sh)RNA along with the respective negative control (NC), which were purchased from Shanghai GeneChem Co., Ltd. A total of 1×10⁵ cells were plated into 6-well plates 24 h prior to stable transfection. Multiplicity of infection (MOI) was determined and the lentivirus was added to the culture medium complemented with the transfection reagent HiTransGA (Shanghai GeneChem Co., Ltd.) with a MOI value of 20–50. After 24 h incubation, the medium was replaced with fresh culture medium containing 2 µg/ml puromycin (Sigma-Aldrich; Merck KGaA) for selection of the stably transfected colonies.

Transient transfection was performed using small interfering (si)RNAs purchased from Shanghai GenePharma Co., Ltd. at a concentration of 20 µM. RNAi-mediated knockdown was performed with the following siRNAs: si-IQGAP3−1, 5′-GGCAGAAACUAGAAGCAUA-3′; si-IQGAP3−2, 5′-GAGCCAACCAGGACACUAA-3′; si-CDC42, 5′-GGACGGAUUGAUUCCACAU-3′; and si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. Cells were transfected with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The subsequent experiments were performed 24–48 h after transfection.

Total RNA was extracted from tissue samples and cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. mRNA was then reverse-transcribed into cDNA using PrimeScript cDNA Synthesis kit (Takara Bio, Inc.) at 37°C for 1 h and then at 85°C for 5 min according to the manufacturer's protocol. qPCR was performed using SYBR-Green Premix Ex Taq II (Takara Bio, Inc.) with a StepOne Plus RT PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: Initial denaturation at 95°C for 5 sec, followed by 40 cycles of annealing at 60°C for 10 sec and an extension at 72°C for 30 sec. β-actin was used as the endogenous control. The primers were designed based on the GeneBank sequences. The primer sequences used were: IQGAP3 forward, 5′-GTGCAGCGGATCAACAAAGC-3′ and reverse, 5′-ACGATGCAACAGGGTACACTG-3′; and β-actin forward, 5′-GAGGCACTCTTCCAGCCTTC-3′ and reverse, 5′-GGATGTCCACGTCACATTC-3′. The comparative threshold cycle method, 2^−ΔΔCq, was used to calculate the relative gene expression level (44).

Cells were harvested and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) with PMSF (1%) and NaF (1%). Protein samples were incubated for 30 min on ice and cell debris were removed by centrifugation at 12,000 × g at 4°C for 15 min. The protein concentration was determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Protein samples (30 µg) were separated by SDS-PAGE (5% stacking gel and 10% separating gel) and transferred to a PVDF membrane (EMD Millipore) using a Bio-Rad Trans-blot system (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk for 1 h at room temperature, the membrane was incubated overnight at 4°C with the primary antibodies. The membranes were then rinsed with TBST (0.1% Tween-20) followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Signals were detected using enhanced chemiluminescence (PerkinElmer, Inc.) with ImageQuant LAS 4000 (GE Healthcare Life Sciences). β-actin was used as the endogenous control. Densitometry analysis was performed using ImageJ version 1.52 g (National Institutes of Health).

The antibodies used were: Rabbit anti-human IQGAP3 (1:1,000; Abcam; cat. no. ab219354), rabbit anti-human CDC42 (1:1,000; Affinity Biosciences; cat. no. DF6322), rabbit anti-human Zinc Finger E-Box Binding Homeobox 1 (ZEB-1; 1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 3396), rabbit anti-human N-cadherin (N-CAD; 1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 13116), rabbit anti-human E-cadherin (E-CAD; 1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 3195), rabbit anti-human Vimentin (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 5741), rabbit anti-human Snail (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 3879), rabbit anti phospho-(p-)AKT (1:1,000; Abcam; cat. no. ab66138), rabbit anti-human AKT (1:1,000; Abcam; cat. no. ab179463), rabbit anti-human PI3K (1:1,000; Abcam; ab182651), rabbit anti-human phosphorylated (p-)mTOR (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 2971), rabbit anti-human mTOR (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 2983), rabbit anti-human Bcl2 (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 2876), rabbit anti-human caspase3 (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 8G10), rabbit anti-human p-ATM Serine/Threonine Kinase (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 5883), rabbit anti-human ATM (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 2873), rabbit anti-human Checkpoint Kinase 2 (CHK2; 1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 2662), rabbit anti-human RAD51 (1:10,000; Abcam; cat. no. ab133534) mouse anti-human Bax (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 2772), mouse anti-human caspase9 (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 9508), rabbit anti-human Pgp (1:1,000; Abcam; cat. no. ab103477) and mouse anti-human β-actin (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 3700).

The secondary antibodies used were: Horseradish peroxidase-conjugated anti-rabbit (1:5,000; Sigma-Aldrich; Merck KGaA; cat. no. A0545) or anti-mouse secondary antibody (1:5,000; Sigma-Aldrich; Merck KGaA; cat. no. A9044).

The proliferative ability of cells was measured using an MTT assay. Each cell line was seeded in quintuplicate into 96-well plates (0.8–1×10³ cells/well) for 0–4 days. At specified time points, 20 µl MTT reagent (Sigma-Aldrich; Merck KGaA) at 5 mg/ml concentration was added to each well, and the cells were incubated for an additional 3.5 h at 37°C. Subsequently, the supernatants were discarded and 100 µl DMSO (Sigma-Aldrich; Merck KGaA) was added to each well. The absorbance at 490 nm was measured using a Varioskan Flash microplate reader (Thermo Fisher Scientific, Inc.).

Cell migration and invasion were analyzed using Boyden chamber-style cell culture inserts, with and without Matrigel (BD Biosciences), respectively. Matrigel was thawed at 4°C and then coated onto the Transwell inserts, after which the gel was allowed to set at 37°C for 1 h. Ovarian cancer cells (2×10⁵ cells) were seeded in the upper chamber of the Transwell inserts (24-well plate; 8-µm pore size; BD Biosciences) with 200 µl serum-free media. The lower chambers were filled with 700 µl culture media containing 10% FBS as the chemoattractant. After 6–48 h of incubation, the cells on the lower surface of the membrane were washed with PBS and fixed in 100% methanol for 15 min at room temperature. Then, cells were stained with 0.1% crystal violet for 20 min at room temperature to quantify migration and invasion. Transwell inserts were observed under a light microscope (magnification, ×10) and cells in 10 random fields were counted.

For the colony formation assay, 500 cells were seeded into each well of a 6-well plate and maintained in media containing 10% FBS at optimum conditions of 37°C with 5% CO₂ for 10–14 days, until the colonies became visible to the naked eye. Colonies were then fixed with 100% methanol at room temperature for 15 min and stained with 0.1% crystal violet at room temperature. Colonies with >50 cells were counted manually under a light microscope (magnification, ×10) for quantification.

Apoptosis was detected using an Annexin V-FITC and propidium iodide (PI) kit (BD Biosciences), according to the manufacturer's protocol. A2780 and HEY cells were transfected with 20 µM si-IQGAP3 or si-NC, and were harvested 48 h after transfection with EDTA-free trypsin, centrifuged at 800 × g for 5 min at room temperature, washed twice with cold PBS, resuspended at a concentration of 1×10⁶ cells/ml and mixed with 100 µl 1X binding buffer. Subsequently, cells were stained with 5 µl Annexin V-FITC and 5 µl PI at room temperature for 15 min in the dark, after which 300 µl 1X binding buffer was added and the cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences) within 1 h. The results were analyzed using FlowJo software version X.0.7 (FlowJo, LLC).

A total of 2×10³ cells/well were seeded in 96-well plates. The A2780 and HEY cells were exposed to olaparib (Selleck Chemicals; cat. no. AZD2281) at various final concentrations (0, 5, 10, 20, 40 and 80 µmol/ml) at 37°C for 36–72 h. Each concentration was repeated in quintuplicate wells. Subsequently, 20 µl 5 mg/ml MTT was added to each well. After incubation for 3.5 h, the medium was replaced with 100 µl DMSO, and cell viability was determined by analyzing the absorbance values at 490 nm on a Varioskan Flash microplate reader (Thermo Fisher Scientific, Inc.).

HEY cells that were stably transfected with IQGAP3-shRNA and the corresponding NC were used for the in vivo experiments. For in vivo experiments, eight female athymic BALB-c nude mice (age, 5 weeks; weight, 20–30 g) were purchased from Nanjing Biochemical Research Institute and housed in a standard pathogen-free condition in individually ventilated cages with HEPA filters at the ambient temperature of 30–31°C and humidity of 50–60% with 12 h light/dark cycle, and adequate access to food and water. For tumor formation assays, 1×10⁷ cells (knockdown or control), resuspended in 200 µl PBS were subcutaneously injected into either side of the axilla.

For metastasis assays, 1×10⁷ cells were intraperitoneally injected individually in the experimental and control groups. After 2–3 weeks, bioluminescence images were captured on an In-vivo imaging system (Kodak 2000 Imager). The mice were euthanized via intraperitoneal injection of 200 mg/kg sodium phenobarbital and the tumors were excised, fixed with 4% paraformaldehyde at room temperature for 48 h, paraffin-embedded and sectioned into 5-µm slices for hematoxylin and eosin staining. The tissue slides were stained with hematoxylin for 5 min and eosin for 10 min at room temperature and observed under a light microscope (magnification, ×4).

GraphPad Prism version 7 (GraphPad Software, Inc.) was used to analyze data. A χ² test was used to analyze the differences in clinical characteristics. Survival analysis was performed using Kaplan-Meier analysis and a log-rank test. An unpaired Student's t-test and a one-way ANOVA were used to determine the statistically significant differences between different groups. Fisher's least significant difference was used for the post-hoc test following ANOVA. Data are presented as the mean ± standard deviation of ≥3 independent experiments. P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression levels of IQGAP3 in healthy fallopian tube and HGSOC tissues were determined using RT-qPCR and western blotting, respectively. The mRNA expression level of IQGAP3 was significantly higher in HGSOC tissues compared with the control samples (Fig. 1D). Furthermore, IQGAP3 protein expression was significantly upregulated in HGSOC tissues compared with the fallopian tubal samples (Fig. 1C).

To examine whether upregulated expression of IQGAP3 was associated with clinical prognosis, IHC staining was performed on 149 HGSOC samples (Fig. 1E). Most positive staining was observed in the cytoplasm and at the cell membrane. Moreover, a high expression of IQGAP3 was observed in 53.02% (79/149) of tissues. Subsequently, the relationship between IQGAP3 and clinicopathological characteristics were assessed (Table I). The patients with a lower expression of IQGAP3 had longer survival times compared with those with higher IQGAP3 expression levels. A log-rank test demonstrated that the upregulated expression of IQGAP3 was significantly associated with overall survival (P=0.0149), as well as progression-free survival (P=0.0044; Fig. 1A).

Survival analysis performed on GEO cohorts using Kaplan-Meier Plotter, showed a significant association between IQGAP3 expression and both overall and progression-free survival (Fig. 1B). In addition, further analysis indicated that the expression of IQGAP3 was associated with several other clinicopathological parameters, including recurrence of the disease (P=0.0065), CA125 levels (P=0.0147) and peritoneal metastasis (P=0.0007; Table I).

Downregulation of IQGAP3 resulted in the reduced proliferation of ovarian cancer cells in vitro. Moreover, two siRNAs, si-IQGAP3−1 and si-IQGAP3−2, were used to silence IQGAP3 in A2780 and HEY cells. MTT assays results identified a significant suppression of the proliferative capacity in the two cell lines following transfection with the siRNAs compared with the NCs (Fig. 2A).

These findings were further assessed in the in vivo experiments, where xenografts of BALB-c nude mice were established with injection of HEY cells stably transfected with sh-IQGAP3 or NC (Fig. 2C). After 3 weeks, the mice were euthanized, imaged on a bioluminescence imaging system, and the tumors were excised and weighed. It was found that there was a significant decrease in tumor size and tumor weight in the sh-IQGAP3 group compared with the NC group (Fig. 2D and E), supporting the in vitro results. Therefore, the results demonstrated the contribution of IQGAP3 to tumor proliferation.

Knockdown of IQGAP3 also significantly reduced colony formation in both A2780 and HEY cell lines (Fig. 2B).

Transwell assays were used to examine the role of IQGAP3 on migration and invasion in vitro. A2780 and HEY cells both had significantly decreased migratory and invasive capacities when IQGAP3 was knocked down compared with the respective NC (Fig. 3A and B).

Furthermore, the underlying mechanism contributing to this increase in tumorigenic features was determined by analyzing EMT-related factors. Knockdown of IQGAP3 had an effect on the expression of several EMT markers (Fig. 4). Silencing of IQGAP3 resulted in downregulation of mesenchymal markers, including ZEB-1, Vimentin, N-CAD and Snail, while the expression of the epithelial marker E-CAD was upregulated. Thus, it was suggested that IQGAP3 induced the migration and invasion of ovarian cancer cells via induction of EMT.

To evaluate the role of IQGAP3 on metastasis of ovarian cancer in vivo, female nude mice were injected intraperitoneally with sh-IQGAP3-HEY cells or their corresponding NCs. Then, 3 weeks after injection, the mice were euthanized and the peritoneal cavities were examined for metastases. Consistent with the in vitro experimental results, mice injected with IQGAP3-silenced cells exhibited significantly lower numbers of metastatic nodules compared with the respective NC group (P<0.05; Fig. 3C). Bioluminescence imaging also identified larger metastatic foci in the control group compared with the knockdown group (Fig. 2E).

The excised metastatic nodules were fixed with formalin and paraffin embedded and 4-µm thick slices were sectioned. Subsequently, the slides were stained using hematoxylin and eosin staining (Fig. S1).

To assess the effects of IQGAP3 knockdown on apoptosis of ovarian cancer cells, Annexin V-FITC/PI dual staining was performed following transfection with si-IQGAP3 or NC. Both A2780 and HEY cells exhibited significantly increased apoptosis following knockdown of IQGAP3 compared with the respective NC group (Fig. 5A and B). These results were further validated by the increased expression of the pro-apoptotic proteins Bax, Caspase 3 and Caspase 9, and decreased expression of Bcl-2 following IQGAP3 knockdown (Fig. 4).

The si-IQGAP3 transfected A2780 and HEY cells were exposed to various concentrations of olaparib (5, 10, 20, 40 or 80 µmol/ml) for 36–72 h, after which, the cell viability was assessed using an MTT assay. Cells transfected with si-IQGAP3 exhibited increased sensitivity to olaparib compared with the respective control group (Fig. 6A). Western blotting results identified the downregulation of the expression levels of Rad51, p-ATM (normalized to total ATM) and CHK2 when IQGAP3 expression was knocked down (Fig. 4). Thus, knockdown of IQGAP3 may have sensitized cells to olaparib by downregulating key factors involved in the DNA damage response.

Phosphoglycolate Phosphatase (Pgp) is a multidrug resistance protein that is localized in the cell membrane and is responsible for extruding several xenobiotics (including chemotherapeutic agents) outside the cells, rendering the cells chemoresistant (45). IQGAP3 knockdown reduced the expression of Pgp, which in turn attenuated chemoresistance (Fig. 4). Therefore, it was speculated that this effect may underlie the enhanced sensitivity of cells towards olaparib following IQGAP3 knockdown.

It has been reported that IQGAP3 is an effector of CDC42 (28). Xu et al (35) also revealed that IQGAP3 may exert its oncogenic function in pancreatic cancer via the regulation of CDC42. To determine whether IQGAP3 was associated with CDC42 in ovarian cancer, the protein expression levels of CDC42 in ovarian cancer cells were assessed by knocking down IQGAP3 expression. It was identified that knockdown of IQGAP3 decreased the expression of CDC42 (Fig. 4).

Therefore, the effects of CDC42 on the cancer cells were assessed. Knockdown of CDC42 expression (Fig. 6B) resulted in a significant decrease in the proliferative potential of HEY cells (Fig. 6C). Furthermore, migration and invasion were inhibited, while apoptosis was enhanced following CDC42 knockdown (Fig. 6D and E). Collectively, these results suggest that IQGAP3 may exert its effects via the regulation of CDC42.