AHNAK suppresses ovarian cancer progression through the Wnt/β-catenin signaling pathway

AHNAK is downregulated in ovarian cancer

Gene Expression Profiling Interactive Analysis illustrated that AHNAK was downregulated in most cancers, including ovarian cancer (Figure 1A). To verify further AHNAK content in ovarian cancer, we collected 30 tumor serous ovarian cancer tissues and neighboring non-malignant tissues. We detected AHNAK content via qRT-PCR along with immunohistochemistry. The results illustrated that AHNAK was downregulated in ovarian cancer tissues (Figure 1B, 1C), indicating that AHNAK could be a tumor suppressor and could play a vital role in ovarian cancer progress.

Figure 1. AHNAK is downregulated in ovarian cancer. (A) AHNAK expression pattern across all tumor samples and matched non-malignant samples. (B) AHNAK expression levels in 30 serous ovarian cancer tissues and paired non-malignant tissues. **p < 0.01. (C) Images illustrating immunohistochemical staining of AHNAK content in two pairs of paired ovarian cancer samples and their neighboring non-malignant tissue. Original magnification, 200X.

AHNAK overexpression represses ovarian cancer cell growth and infiltration in vitro

To determine the significance of AHNAK in the progression of ovarian cancer, AHNAK was overexpressed in ovarian cancer cells (Figure 2A). A cell proliferation assay was conducted, and the results demonstrated that AHNAK overexpression suppressed the cell proliferation of ovarian cancer cells (Figure 2B). Moreover, the CCK-8 data illustrated that AHNAK overexpression dramatically repressed the proliferation of ovarian cancers (Figure 2C). Furthermore, we conducted a Transwell assay to assess the function of AHNAK in infiltration of ovarian cancer cells. The data illustrated that elevated content of AHNAK repressed the infiltration of ovarian cancer cells (Figure 2D). To explore whether AHNAK overexpression triggered MET, we assayed the content of the epithelial, as well as mesenchymal biomarkers. Western blotting revealed that AHNAK overexpression elevated E-cadherin levels and suppressed N-cadherin and vimentin levels (Figure 2E).

Figure 2. Elevated AHNAK content represses ovarian cancer cell proliferation along with infiltration in vitro. (A) qRT-PCR illustrated that AHNAK was successfully overexpressed. (B) Cell proliferation analysis was performed. Cell numbers were evaluated at 24 h, 48 h, and at 72 h after incubation by Coulter Counter (Beckman Coulter, USA). (C) CCK-8 assay was performed. (D) Transwell assays were performed (left), after which infiltrating cells were quantified by the Image J software (right). (E) Western blotting assessment of the levels of indicated epithelial and mesenchymal markers. Data are presented as the mean ± SD for n=3, *p < 0.05, **p < 0.01.

AHNAK overexpression suppresses ovarian cancer tumor growth along with migration in vivo

To further assess the biological impacts of AHNAK on ovarian cancer in vivo, we established a mouse xenograft model. We found that AHNAK overexpression remarkably diminished tumor growth (Figure 3A), as well as lung metastasis, as illustrated in Figure 3B. The results indicated that AHNAK overexpression represses tumor growth along with migration in ovarian cancer.

Figure 3. Elevated AHNAK content suppresses ovarian cancer tumor growth along with migration in vivo. (A) The tumors from the mouse xenograft model (n = 3 per group) are shown (left). The weights of the tumors are summarized (right). (B) HE-stained sections derived from lung metastatic nodules of the mouse xenograft model (n = 3 per group) are illustrated (left). The number of lung nodules was quantified (right). Original magnification: 40X and 200X, **P < 0.01.

AHNAK represses ovarian cancer progression through the Wnt/β-catenin cascade

On the basis of the results of the above experiments, we further assessed the possible molecular mechanisms through which AHNAK exerts its functions in ovarian cancer. It has been documented that the aberrantly activated Canonical Wnt cascade participates in development of cancer [10], and Wnt–β-catenin is correlated with cancer metastasis [11]. Therefore, we investigated whether AHNAK functioned via the Canonical Wnt cascade. The Canonical Wnt axis inhibitor Dkk1 was employed to confirm the function of AHNAK in activating canonical Wnt cascade. The content of wnt-1, β-catenin, and β-actin proteins in si-AHNAK cells was remarkably elevated in contrast with the controls, and Dkk1 effectively repressed this effect. Western blotting (Figure 4A) and qRT-PCR (Figure 4B) revealed that AHNAK knockdown increased the contents of Canonical Wnt cascade markers, indicating that AHNAK functioned partly via modulating the Canonical Wnt axis.

Figure 4. AHNAK represses ovarian cancer progress by targeting the Wnt/β-catenin cascade. (A) Western blotting evaluation of the contents of Canonical Wnt cascade markers. The Western blotting image in the right panel exhibits that the Wnt inhibitor, DKK1, suppressed Wnt signaling of AHNAK-siRNA activated signal in OV119 and A2780 cells. (B) qRT-PCR assays of the contents of Canonical Wnt cascade markers. Data are presented as the mean ± SD for n=3, **p < 0.01.

Gene expression of AHNAK in GEPIA

GEPIA (http://gepia.cancer-pku.cn/index.html), a web server for assessing RNA expression data for 9,736 cancers and 8,587 non-malignant samples from TGGA along with the Genotype-Tissue Expression projects. The expression profile of AHNAK in different cancers was explored in GEPIA.

Clinical samples and immunohistochemistry

Thirty paired serous ovarian tumor and neighboring non-malignant tissues were acquired from The First Affiliated Hospital of the University of South China. The immunohistochemistry (IHC) of AHNAK staining (1:250, Abcam, USA) was performed as described previously [5]. Certified pathologists evaluated the staining in a blinded manner.

Cell growth and transfection

Ovarian cancer OV119 and A2780 cell lines were obtained from the American Type Culture Collection (USA) and were grown as described by the manufacturer. Cells were validated to be mycoplasma infection-free, and the authenticity was confirmed via DNA fingerprinting.

For AHNAK overexpression experiments, an AHNAK-expressing lentivirus vector (EX-V0190-Lv122) as well as control plasmid (EX-NEG-Lv122) were provided by GeneCopoeia (USA). The Lenti-Pac^™ HIV Expression Packaging Kit (Cat No, GeneCopoeia) was employed to generate lentiviruses. Lentiviral cell infections were performed using Lipofectamine 2000 (Cat No, Invitrogen, USA) and screened with puromycin (2 μg/mL) to obtain AHNAK-expressing cells. For AHNAK knockdown experiments, cells were inserted with si-AHNAK (Santa Cruz Biotechnology, sc-97060) via transfection as described by the manufacturer. A nontargeting siRNA was used as control. Then, the cells were grown and utilized in the subsequent assays.

Quantitative RT-PCR (qRT-PCR) analysis

RNA extraction was performed using the TRIzol reagent (Invitrogen). Denaturing agarose gel was employed to check RNA integrity via electrophoresis. PrimeScript^TM RT Master Mix as well as SYBR^® Premix Ex Taq^TM II (Cat No, Takara, Japan) were used to perform qRT-PCR using the Bio-Rad CFX96 PCR System (USA). Primers used in this study were synthesized by Invitrogen and they are shown in Supplementary Table 1. β-actin value served as the normalization standard and calculated by the 2^-ΔΔCt approach. Each reaction was repeated in triplicates.

Cell proliferation assay

OV119 and A2780 cells (2 × 10⁴) were planted, and we counted the numbers of cells at 24 h, 48 h, and at 72 h post incubation using the Coulter Counter (Beckman Coulter, USA).

CCK-8 assay

OV119 and A2780 cells (1 × 10³) inoculated at 37° C for 24h prior to transfection. Then, cells were incubated for another 48 h after which the CCK-8 solution (Cat No, Dojindo Laboratories, Japan) was introduced. Cells were incubated for 2 h at 37° C after which absorbance at 450 nM was read using a microplate reader.

Transwell assay

OV119 and A2780 cells (1 × 10⁴) were cultured, and a medium supplemented with 10% FBS introduced in the lower compartment (BD Biosciences, USA) as a chemoattractant. Afterwards, we fixed the cells with methanol, followed by 0.1% crystal violet staining, and counting.

Wnt treatment and western blot assay

OV119 and A2780 cells were cultured with or without the addition of the Wnt–β-catenin cascade inhibitor Dkk1 (100 ng/ml, R&D Systems). Proteins of OV119 and A2780 were extracted, and then the concentration determined, fractionated by SDS-PAGE gel (10%) and transfer-embedded to PVDF membranes (Millipore, USA). Then, membranes were blocked with 5% skim milk for 1 h at room temperature. Then the membranes were inoculated with primary antibodies against E-cadherin (Cell Signaling Technology, USA; 1:1000), N-cadherin (Cell Signaling Technology; 1:1000), vimentin (Cell Signaling Technology; 1:1000), wnt-1 (Abcam, USA; 1:1000), β-catenin (Cell Signaling Technology; 1:1000), as well as c-myc (Cell Signaling Technology; 1:1000). Then, an HRP-labeled secondary antibody (Cell Signaling Technology) was added. The anti-β-actin antibody (Affinity, USA; 1:1000) was used as the control.

Mouse xenograft model

The animal study was performed as previously described [25, 26]. OV119 and A2780 cells were stably transfected with AHNAK or vector and suspended in PBS at 1 × 10⁷ cells/ml, respectively. Then, cells (2 × 10⁶) were subcutaneously inoculated into 4 week old female BALB/c nude mice using a 1-ml injector. (n = 3 per group to provide a power of 90% for a remarkable level of 0.05 with a two-tailed t-test). After 4 weeks, mice were anesthetized after which xenograft tumors were excised and tumor weights determined.

For lung metastasis experiments, OV119 and A2780 cells (1 × 10⁵) were administered through the tail vein by using a microsyringe (n = 3 per group to provide a power of 90% for a remarkable level of 0.05 with a two-tailed t-test). Cells (2 × 10⁶ cells/ml) were dispersed in PBS. After 8 weeks, mice were anesthetized after which lungs were excised. The number of lung macro metastatic nodules were counted and verified by hematoxylin and eosin (HE) staining, which was explored via certified pathologists in a blinded manner.

Statistical analysis

Statistical analyses were implemented using the SPSS 19.0 software. Between group comparisons were performed using t-tests. Data are presented as the mean ± SD for three independent experiments, unless otherwise stated. Differences were considered statistically significant when p ≤ 0.05.

Data accessibility statement

The data supporting the findings in this study are available from the corresponding author upon reasonable request.

Ethics committee approval and patient consent

Ethical approval for this study was obtained from the Ethics Committee of The First Affiliated Hospital of the University of South China. Experiments were performed according to the ethical standards formulated in the Declaration of Helsinki. Informed consents were obtained from all patients. The use of animals in this study was approved by the Institutional Research Ethics Committee of The First Affiliated Hospital of the University of South China and performed according to institutional guidelines.