Angiotensin II type 2 receptor–interacting protein 3a inhibits ovarian carcinoma metastasis via the extracellular HMGA2-mediated ERK/EMT pathway

Abstract

Local migration and long-distance metastasis is the main reason for higher mortality of ovarian cancer. Microtubule-associated tumor suppressor 1/angiotensin II type 2 receptor–interacting protein is associated with tumor initiation and progression and exerts anti-tumor effects. High mobility group AT-hook 2 is overexpressed in majority of metastatic carcinomas, which contributes to carcinomas metastasis through Snail-induced epithelial-to-mesenchymal transition signal pathway. The purpose of this study was to investigate the signal pathway of microtubule-associated tumor suppressor 1/angiotensin II type 2 receptor–interacting protein–mediated anti-tumor effects. Our data observed that ovarian carcinoma cells exhibited lower expression of angiotensin II type 2 receptor–interacting protein 3a and higher expression of high mobility group AT-hook 2 compared to normal ovarian cells. Restoration of angiotensin II type 2 receptor–interacting protein 3a expression in ovarian carcinoma cells inhibited high mobility group AT-hook 2 expression and exhibited anti-proliferative effects. In addition, angiotensin II type 2 receptor–interacting protein 3a treatment suppressed the phosphorylation of epithelial-to-mesenchymal transition and extracellular signal–regulated kinase in ovarian carcinoma cells. We also observed that angiotensin II type 2 receptor–interacting protein 3a restoration downregulated expression of Snail, E-Cadherin, N-Cadherin, and Vimentin in ovarian carcinoma cells, whereas angiotensin II type 2 receptor–interacting protein 3a knockdown enhanced the phosphorylation of extracellular signal–regulated kinase and epithelial-to-mesenchymal transition. In vivo assay indicated that angiotensin II type 2 receptor–interacting protein 3a inhibited ovarian tumor growth and elevated survival of tumor-bearing immunodeficient mice. Tumor histological analysis indicated that Snail, E-Cadherin, N-Cadherin, and Vimentin expression levels were downregulated via decreasing high mobility group AT-hook 2 expression. Furthermore, upregulation of angiotensin II type 2 receptor–interacting protein 3a impaired the phenotype of extracellular signal–regulated kinase and epithelial-to-mesenchymal transition in ovarian carcinoma cells and tumor tissues. Taken together, angiotensin II type 2 receptor–interacting protein 3a presents potential in suppressing the proliferation and aggressiveness of ovarian carcinoma cells through the high mobility group AT-hook 2–mediated extracellular signal–regulated kinase/epithelial-to-mesenchymal transition signal pathway.

Introduction

Worldwide, ovarian cancer is one of the most common tumors and shows the highest incidence and mortality among all gynecologic malignancies. ¹ Ovarian cancer is a diverse and gnomically complex disease that presents higher morbidity and mortality for it is often diagnosed at a late stage in clinic. ² Previous study reported that the incidence rate of ovarian cancer is increasing and presents a rapid expansion trend in the world. ³ Women patients with ovarian cancer reveal a continued symptom of depression and anxiety for cancer survivors who had undergone chemotherapy treatment by meta-analysis of prevalence rates. ⁴ Therefore, the effective agents and protocols are urgently needed to improve the high rate of occurrence and metastasis for patients with ovarian cancer after curative resection. ⁵

Currently, a number of research studies demonstrated that extracellular signal–regulated kinase (ERK) and epithelial-to-mesenchymal transition (EMT) are two key processes in tumor biological function and clinical aggressiveness during tumor carcinogenesis, initiation, and progression.^6,7 Previous study has identified that EMT/ERK can promote growth, invasion, and metastasis of cancer, which may play important role in multiple more aggressive tumor cells by endowing cells with a more motile and invasive potential. ⁸

High mobility group AT-hook 2 (HMGA2) is a chromatin remodeling factor that can change the chromatin architecture to regulate the activity of transcriptional enhancers. ⁹ Previous study has showed that HMGA2 evidences as an architectural transcription factor, which is related with progression and prognosis of many malignant cancers. ¹⁰ In addition, HMGA2 is highly expressed in most malignant epithelial carcinoma, and HMGA2 overexpression in transgenic mice causes tumorigenesis resulting in tumor growth and development. ¹¹ Furthermore, overexpression of HMGA2 promoted tongue cancer metastasis through EMT pathway.^12,13 In this study, we investigate the regulatory mechanism of HMGA2 in ovarian carcinoma both in vitro and in vivo.

Microtubule-associated tumor suppressor 1 (MTUS1) is identified as a tumor suppressor gene encoding a family of angiotensin II type 2 (AT2) receptor–interacting proteins (ATIPs). ¹⁴ The ATIP polypeptides exhibits distinct motifs in the amino-terminus for localization to the cytosol, nucleus, or cell membrane, suggesting that MTUS1 gene products may be involved in a variety of intracellular functions in an AT2-dependent and AT2-independent manner.^15,16 Downregulation of the MTUS1 gene, particularly ATIP1 and ATIP3a, has been found in the majority of epithelial cancer cells. ¹⁷ A report showed that 8p22 MTUS1 gene ATIP3 is a novel anti-mitotic protein that shows indicative role in invasive breast carcinoma of poor prognosis. ¹⁸ Another study indicated that downregulation of tumor suppressor MTUS1/ATIP is associated with enhancement of proliferation, poor differentiation, and poor prognosis in oral tongue squamous cell carcinoma (TSCC). ¹⁹ Furthermore, restoration of ATIP3 expression led to G1 arrest, apoptosis, and inhibited TSCC cell proliferation and invasion. ²⁰ Although these reports have explained the function of MTUS1, the molecular mechanism of MTUS1 still keeps unknown.

This study investigated the role and mechanism of ATIP3a in ovarian carcinoma. The anti-proliferative effects of ATIP3a in ovarian carcinoma were investigated in ovarian carcinoma cells. The inhibitory effects of ATIP3a in the migration and invasion of ovarian carcinoma cells and the involvement of signaling pathway were also investigated. Our data indicate that the inhibitory effects of ATIP3a on proliferation, migration, and invasion of ovarian carcinoma are mediated by ERK/EMT signaling pathway.

Materials and method

Cells culture and reagents

Ovarian cancer tissues from patients were collected with written informed consent. HOEC-1, SKOV3, CAOV-3, and A2780 cells were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA). All cells were cultured in a 37°C humidified atmosphere of 5% CO₂.

Lactate dehydrogenase assay

The ovarian tumor cells were cultured until monolayer cells reached 90%, and then media was removed. The ovarian tumor cells were washed three times and subsequently incubated with Triton X-100 (1%) for 30 min. Lactate dehydrogenase (LDH) activity in the lysates was performed by using the Promega CytoTox 96 assay kit. The detailed procedures were conducted according to the manufacturer’s instructions.

Cell invasion and migration assays

SKOV3, CAOV-3, and A2780 cells were treated with ATIP3a or transfected with small interfering RNA (siRNA)-ATIP3a with siRNA-vector as control. For migration assay, HGC-27 and cells were treated with ATIP3a or transfected with siRNA-ATIP3a and incubated for 96 h by using a control insert (BD Biosciences) instead of a Matrigel Invasion Chamber. For invasion assay, cells treated with ATIP3a or transfected with siRNA-ATIP3a were suspended as a density of 1 × 10⁵ in 500 µL in serum-free DMEM. The cells were then subjected to the tops of BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer’s instructions. The tumor cells invasion and migration were counted in at least three randomly stain-field microscope every membrane.

Real-time quantitative polymerase chain reaction

Total RNA was obtained from SKOV3, CAOV-3, and A2780 cells using RNAeasy Mini Kit (Qiagen, Gaithersburg, MD). A total of 1 µg total RNA was transcribed into complementary DNA (cDNA) by using the reverse transcription kit (Qiagen). The synthetic cDNA was confirmed by electrophoresis for its quality. Subsequently, the synthetic cDNA (10 ng) was subjected to a real-time quantitative polymerase chain reaction (RT-qPCR) (Bio-Rad, Hercules, MD) with SYBR Green Master Mix system (Applied Biosystems, Foster City, CA, USA). Difference in messenger RNA (mRNA) expression changes were calculated by 2^−ΔΔCt. All the forward and reverse primers were synthesized by Invitrogen. The results are expressed as the n-fold way compared to control.

Immunohistochemical staining

Immunohistochemical staining was performed by an avidin–biotin–peroxidase technique. Paraffin-embedded tumor tissue sections were prepared and epitope retrieval was performed for further analysis. The paraffin sections were subjected to hydrogen peroxide (3%) for 10–15 min, which subsequently were blocked by a regular blocking solution for 10–15 min at 37°C. Finally, the sections were incubated with anti-ATIP3a, anti-E-Cadherin, anti-N-Cadherin, anti-Vimentin, anti-ERK, and anti-TUNEL/DAPI, at 4°C for 12 h after blocking. All sections were washed three times and incubated with secondary antibodies for 1 h at 37°C and were observed by six random views in the microscope.

Western blot

A2780 cells were treated by ATIP3a and homogenized in lysate buffer containing protease-inhibitor and were centrifuged at 8000 r/min at 4°C for 10 min. The supernatant of mixture were used for the analysis of purpose protein. For the detection of purpose protein, transmembrane protein was extracted by using Transmembrane Protein Extraction Kit (Qiagen) according to the manufacturer’s instructions. Sodium dodecyl sulfate (SDS) assays were performed as previous descript. ²¹ For western blotting, primary antibodies were added after blocking (5% skimmed milk) for 1 h at 37°C and then incubating with secondary antibodies 24 h at 4°C. The results were visualized by using chemiluminescence detection system.

Animal experiments

Specific pathogen-free (SPF) female BALB/c (6 weeks old) nude mice were purchased from Slack Experimental Animals Co., Ltd (Slack, Shanghai, China). All animals were fed under pathogen-free conditions. A total of 1 × 10⁶ density of A2780 cells was injected into the ovarian of female BALB/c nude mice in a total volume of 200 µL. Therapy for tumor-bearing mice by ATIP3a was initiated when tumor diameters reached 4–6 mm on day 6 after tumor inoculation. Mice with ovarian carcinoma were randomly divided into two groups (n = 40 in each experimental group) and injected intratumorally (10 mg/kg) with the same volume phosphate-buffered saline (PBS) as control. The treatment was continued 10 times at intervals of every 2 days of injection. Tumor diameters were recorded once in every 2 days and tumor volume was calculated by using the formula: 0.52 × smallest diameter² × largest diameter. The tumor metastasis was evaluated by tumor occurrence in the other subcutaneous sites.

Flow cytometer analysis apoptosis

A2780 cells were cultured in DMEM medium supplied 10% FBS for 48 h. A2780 cells were treated with ATIP3a with PBS as control for 48 h. All cells were subsequently treated by cisplatin for 12 h. The apoptosis of suspended cells was analyzed by flow cytometer as described in a previous report. ²²

Statistical methods

All presented data were reported as means and standard error of the mean (SEM). Unpaired data were analyzed by Student’s t test. Comparisons of data between multiple groups were analyzed by analysis of variance (ANOVA). *p < 0.05 and **p < 0.01 were considered statistically significant.

Results

ATIP3a inhibits the proliferation and aggressiveness of ovarian cancer cells through regulating HMGA2 expression

Lower expression of ATIP3a has been reported in various human cancers.^23,24 We showed that ovarian tumor tissue presented lower ATIP3a expression compared to normal ovarian tissue (Additional file 1—Supplementary Material). This study also investigated the ATIP3a expression in ovarian cancer cells determined by western blotting and RT-qPCR to quantify the expression level. The results revealed that both mRNA and protein levels of ATIP3a protein were lowly expressed in ovarian cancer cell lines compared to human ovarian surface epithelial cells (HOEC-1) (Figure 1(a) and (b)). To investigate the relationship between ATIP3a and HMGA2, we analyzed HMGA2 expression in ovarian cancer cell lines. Our data showed that upregulation of the HMGA2 in mRNA and protein levels were significantly higher in ovarian cancer cell lines (Figure 1(c) and (d)). In addition, CAOV-3 and A2780 cells were treated with ATIP3a, resulting in a statistically significant suppression of HMGA2 expression and cell growth compared to control (Figure 1(e) and (f)). Furthermore, migration and invasion of ovarian cancer cells were also inhibited after restoration of ATIP3a (Figure 1(g) and (h)). Taken together, these data indicate that ATIP3a could significantly inhibit the proliferation and aggressiveness of ovarian cancer cells through upregulating HMGA2 expression.

Figure 1.

ATIP3a inhibits the proliferation and aggressiveness of ovarian cancer cells through downregulating HMGA2 expression. (a) mRNA and (b) protein expression levels of ATIP3a in ovarian cancer cell lines. (c) Analysis of mRNA and (d) protein expression levels of HMGA2 in ovarian cancer cell lines. (e) Changes of mRNA levels of HMGA2 in ovarian cancer cell lines after reconstitution of ATIP3a. (f) Analysis of growth of cancer cell lines after reconstitution of ATIP3a. Analysis of (g) migration and (h) invasion of cancer cell lines after reconstitution of ATIP3a.

ATIP3a regulates the HMGA2-induced ERK/EMT pathway in ovarian cancer cells

In order to study the mechanism of ATIP3a-meidated anti-proliferative effects in ovarian cancer cells, we analyzed ERK/EMT signaling pathway. As shown in Figure 2(a), the ATIP3a treatment led to significant downregulation of HMGA2, Snail, E-Cadherin, N-Cadherin, Vimentin, and ERK both in expression and in phosphorylation (Figure 2(a)). However, knockdown of endogenous ATIP3a by siRNA markedly increased the protein levels of HMGA2, Snail, E-Cadherin, N-Cadherin, Vimentin, and ERK (Figure 2(b)). In addition, we also observed significant decreasing expression of E-Cadherin and N-Cadherin in A2780 cells determined by immunofluorescence (Figure 2(c)). Tumor cell growth and aggressiveness were also promoted after silenced endogenous ATIP3a (Figure 2(d) and (e)). However, restoration of HMGA2 canceled inhibition of knockdown of endogenous ATIP3a in inactivity of ERK/EMT signal pathway (Figure 2(f)). Furthermore, statistically significant reduced number of tumor clones was observed in addition to ATIP3a and decreased levels of tumorigenicity after restoration of HMGA2 (Figure 2(g) and (h)) Taken together, these results confirm the ATIP3a-inhibting function of ERK/EMT, which is associated with suppressed proliferation and invasive activities of metastatic ovarian cancer cells.

Figure 2.

ATIP3a suppresses the ERK/EMT pathway induced by HMGA2 in ovarian cancer cells. (a) Analysis of molecules in HMGA2-induced ERK/EMT pathway in ATIP3a-treated A2780 cells. (b) HMGA2-induced ERK/EMT signaling pathway was promoted after silencing ATIP3a. (c) Immunofluorescence analysis of E-Cadherin and N-Cadherin in ATIP3a-treated A2780 cells. (d) Growth and (e) aggressiveness of ovarian cancer cell lines were enhanced after silencing ATIP3a. (f) Analysis of EMT and ERK pathway in A2780 cells after silencing ATIP3a. (g) Inhibition of proliferation and (h) carcinogenicity induced reconstitution of ATIP3a in A2780 cells.

ATIP3a reconstitution inhibits HMGA2-induced ERK/EMT phenotype in ovarian cancer cells

In Figure 3(a), our data showed that HMGA2, Snail, and Cadherin were highly expressed in ovarian cancer tissues, whereas lowly expressed in cancer tissues. To further illustrate the relationship between ATIP3a and HMGA2, the correlation between ATIP3a and HMGA2 was analyzed by Pearson analysis and the result indicated that there is a positive correlation between them (R² = 0.8876, p < 0.001; Figure 3(b)). The data showed that ATIP3a reconstitution inhibited ERK and EMT (Figure 3(c)). We also found that ATIP3a reconstitution promoted apoptotic sensibility of ovarian cancer cells induced by cisplatin (Figure 3(d)). By fluorescence microscopy analysis, ATIP3a reconstitution showed a significant decrease of fluorescence of HMGA2 and supplement of HMGA2 significantly almost abolished the inhibitory effects of ATIP3a reconstitution (Figure 3(e)). Importantly, our results showed that downregulation of ERK/EMT was observed after ATIP3a reconstitution for 48 h (Figure 3(f)). Taken together, these data indicate that ATIP3a reconstitution inhibits HMGA2-induced ERK/EMT phenotype in ovarian cancer cells.

Figure 3.

Inhibition of HMGA2-induced ERK/EMT phenotype in ovarian cancer cells after reconstitution of ATIP3a. (a) Analysis of HMGA2, Snail, Cadherin, and ATIP3a expression in ovarian cancer tissues. (b) Analysis of the correlation between ATIP3a and HMGA2 determined by Pearson analysis. (c) Downregulation of Ras, Raf, Snail, and Cadherin in ATIP3a-treated A2780 cells. (d) ATIP3a promoted apoptosis of A2780 cells. (e) ATIP3a elevated HMGA2 expression in A2780 cells. (f) Analysis of the function of ATIP3a and HMGA2 in ERK/EMT signaling pathway.

ATIP3a reconstitution inhibits ovarian tumor growth via ERK/EMT signaling pathway in vivo

In order to investigate ATIP3a-induced ERK/EMT signaling pathway and whether ATIP3a reconstitution affected on ovarian tumor growth, we studied the inhibitory effects on tumor growth in xenograft mice in vivo. We found that ATIP3a reconstitution suppressed the tumor growth in A2780-bearing xenografts (Figure 4(a)). We observed that ATIP3a reconstitution increased the apoptotic body and apoptosis rate in tumors in mice (Figure 4(b)). In addition, transcription factors of ERK/EMT signaling pathway were downregulated in tumors after ATIP3a reconstitution (Figure 4(c)). The expression of HMGA2 was downregulated in tumor cells and tissues after ATIP3a reconstitution in vivo analysis (Figure 4(d)). Notably, tumor metastasis was significantly inhibited after ATIP3a reconstitution (Figure 4(e)). In addition, statistical analysis employing the Mann–Whitney test showed significant differences of microvascular density among the mice harboring ATIP3a reconstitution and PBS-treated mice on day 30 (Figure 4(f)). Collectively, our findings suggest that ATIP3a reconstitution inhibits ovarian tumor growth via ERK/EMT signaling pathway in vivo.

Figure 4.

Inhibition of tumor growth and metastasis through targeting ERK/EMT in A2780-bearing mice. (a) Tumor growth was analyzed after reconstitution of ATIP3a or PBS treatment in 30-day short-term observation. (b) Apoptotic rate and bodies from experimental mice were analyzed after treatment of ATIP3a. (c) ERK/EMT signaling pathway was downregulated after reconstitution of ATIP3a. (d) The expression of HMGA2 was downregulated in tumor cells and tissues after ATIP3a reconstitution in vivo analysis. (e) Metastatic dissemination in the omentum, diaphragm, mesentery, and peritoneal wall was assessed by counting metastatic colonies. (f) Blood vessel density in tumors was analyzed by observing numbers of angiogenesis.

Discussion

Aggressiveness of cancer cells plays important roles in tumor progression by acquiring the ERK/EMT phenotype. ²⁵ Therefore, therapies targeting ERK/EMT pathways are believed to be promising therapeutic strategies for the majority of human carcinomas. In this study, we reported that ATIP3a expression is insufficient and HMGA2 expression is higher in ovarian cancer cells and tumor tissues. HMGA2 overexpression induced ERK/EMT signaling pathways that further promoted migration and invasion of ovarian cancer cells. Reconstitution of ATIP3a efficiently inhibited growth and aggressiveness both in vitro and in vivo. These findings suggest that ATIP3a inhibits ovarian carcinoma metastasis via the extracellular HMGA2-mediated ERK/EMT pathway, which may be a potential anti-cancer agent in the treatment of ovarian carcinoma.

HMGA2 is frequently overexpressed in cancer cells during embryogenesis, which orchestrates transcription activity by modulating chromatin structure through binding to DNA sequences.^26,27 Previous study has been reported that downregulation of HMGA2 led to inactivate the Snail-induced EMT processes in TSCC, which further resulted in tumor regression and inhibition of metastasis in various cancers. ²⁸ However, the upstream regulatory mechanism of HMGA2 still remains unknown. In this study, our data are consistent with previous reports and we demonstrated that HMGA2 is upregulated in ovarian cancer lines and tissues, which positively associated with proliferation and metastasis via activating the EMT processes.^12,29 Notably, our finding indicates that ATIP3a treatment can downregulate HMGA2 expression and further inhibit growth, progression, and development of ovarian tumors. We present that ATIP3a reconstitution inhibits HMGA2 expression, resulting in inhibition of growth and metastasis via inactivating the ERK/EMT processes.

ATIP3a polypeptides contain a nuclear localization signal in the amino-terminus and results have found that restoring ATIP3 expression could lead to suppression of proliferation, clonogenicity, and anchorage-independent growth in carcinoma cells. ²³ A study has indicated that ATIP3a restoration alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells undergoing active mitosis. ³⁰ ATIP3a is also reported to involve ERK signaling pathway that belongs to the family of MAPKs and is downregulated after reconstitution of ATIP3a. ²⁴ In addition, EMT pathway is also associated with ATIP3a-mediated inhibition of tumor growth and progression. ³¹ In this study, our findings show that reconstitution of ATIP3a inhibits ovarian cancer growth and promotes apoptosis induced by cisplatin. Signaling molecules in ERK/EMT pathway are markedly downregulated in the treatment of ATIP3a that contributes to ATIP3a-induced suppression of proliferation, migration, and invasion of ovarian carcinoma cells and tissues.

In conclusion, the beneficial outcomes of reconstitution of ATIP3a on inhibition of tumor growth and increasing survival rate have been observed in a 30-day period. The findings exert notable anti-proliferative effects and inhibit the migration and invasion of ovarian carcinoma cells via regulation of the ERK/EMT pathway. These data suggest that the clinical relevance of reconstitution of ATIP3a for cancer therapy needs further development in prospective larger scale investigation, which may be a novel anti-cancer agent for ovarian carcinoma therapy.