Disabled homolog 2 is required for migration and invasion of prostate cancer cells

Abstract

Disabled homolog 2 (DAB2) is frequently deleted or epigenetically silenced in many human cancer cells. Therefore, DAB2 has always been regarded as a tumor suppressor gene. However, the role of DAB2 in tumor progression and metastasis remains unclear. In this study, DAB2 expression was upregulated along with human prostate cancer (PCa) progression. DAB2 overexpression or knockdown effects in LNCaP and PC3 cell lines were verified to address the biological functions of DAB2 in PCa progression and metastasis. LNCaP and PC3 cell lines were generated from human PCa cells with low and high metastatic potentials, respectively. The results showed that DAB2 shRNA knockdown can inhibit the migratory and invasive abilities of PC3 cells, as well as the tumorigenicity, whereas DAB2 overexpression enhanced LNCaP cell migration and invasion. Further investigation showed that DAB2 regulated the cell migration associated genes in PC3 cells, and the differential DAB2 expression between LNCaP and PC3 cells was partly regulated by histone 4 acetylation. Therefore, DAB2 may play an important role in PCa progression and metastasis.

Keywords

DAB2 / prostate cancer / migration / invasion / acetylation

Introduction

Prostate cancer (PCa) is one of the most frequently observed carcinomas among males, and the prognosis of advanced PCa appears to be poor [ 1]. Androgen suppression is usually performed as an effective treatment for patients with late-stage PCa. Unfortunately, many patients ultimately develop androgen-independent PCa over time, followed by distant metastasis, which is the most common cause of death among PCa patients [ 2]. Therefore, determination of the molecular mechanism underlying PCa metastases is very urgent.

LNCaP and PC3 cell lines are the most commonly used tools in PCa research. PC3 cells demonstrate high metastatic potential, whereas LNCaP cells exhibit low metastatic potential [ 3]. PC3 cells are ideally used to investigate the biochemical changes in advanced PCa cells. Interestingly, a high expression of disabled homolog 2 (DAB2) in PC3 cells was observed in a previous work [ 4]. DAB2 gene, initially named as DOC2 (deletion in ovarian carcinoma 2) [ 5], is known as one of the two mammalian orthologs of the Drosophila disabled gene. DAB2 encodes a phosphoprotein and has been characterized as an endocytic adaptor, which plays an important role in endocytosis and stem cell specification [ 6]. Knockout of Dab2 caused endoderm disorganization in E5.5 mouse embryos [ 7]. As an important adaptor molecule, DAB2 is required for regulating many signaling pathways, such as Ras/MAPK signaling and Wnt signaling pathway [ 8]. DAB2 is also a target gene of transforming growth factor-β (TGF-β) and participates in epithelial-to-mesenchymal transition (EMT) [ 9], a process in which cells undergo a switch from a polarized epithelial phenotype to a highly motile fibroblastic or mesenchymal phenotype. EMT enables tumor cells to acquire migratory and invasive properties [ 10], indicating that DAB2 may affect tumor progression.

However, DAB2 has been regarded as a tumor suppressor gene because of its low expression and deletion in various tumor cells [ 11]. In 1994, Mok et al. reported that DAB2 was downregulated or absent in ovarian carcinoma cell lines compared with normal ovarian epithelial cells [ 12]. Subsequent research showed that enforced DAB2 overexpression can significantly reduce the growth rate of ovarian carcinoma cells, as well as their tumor formation ability in nude mice [ 13]. By contrast, Chao et al. indicated that DAB2 suppression by miR-187 can inhibit cell migration, whereas DAB2 upregulation can promote EMT, leading to augmentation of the migratory and invasive abilities of tumor cells [ 14]. Two other groups also reported that DAB2 promotes and spreads tumor cell migration [ 15, 16]. Therefore, DAB2 may serve multiple functions in tumor transformation and progression.

The present study revealed that DAB2 expression was upregulated along with human PCa progression, and DAB2 was required for the invasion and migration of PCa cells, suggesting that DAB2 may play important roles in PCa progression and metastasis.

Materials and methods

Clinical samples

PCa tissue was collected from patients at Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine after obtaining informed consent.

Antibodies and inhibitors

The following antibodies and inhibitors were purchased: anti-DAB2 (1:1000, sc-13982, Santa Cruz Biotechnology), anti-β-actin antibody (1:5000, sc-47778, Santa Cruz Biotechnology), and histone acetyl transferase (HAT) inhibitor C646 (25 µM, S7152, Selleck).

Cell culture

Human PCa cell lines, PC3 and LNCaP, were purchased from the Chinese Academy of Sciences Cell Bank of Type Culture Collection. PC3 was maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). LNCaP and 293T cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% FBS (Gibco). All cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂.

Plasmid construction, virus preparation, and infection

Full-length cDNA of DAB2 was subcloned into MigR1-GFP vector. shRNA recombinant plasmids were constructed using PLSLG vector, and the DAB2 specific shRNA sequences were listed as reported by Orlandini et al. [ 17]: DAB2-shRNA22, 5′-GTC CAT ACA GAA TGG CGT AAA-3′, DAB2-shRNA47, and 5′-GTA TCT GAA AGA GAA CAG AAC-3′. PLSLG-DAB2-shRNA plasmids were co-transfected into 293T cells with PMD2.G and PSPAX2 by using Lipofectamine 2000 (Invitrogen) to prepare lentiviruses carrying DAB2-shRNA. Culture supernatants were collected and concentrated 48 h after transfection. For the infection process, viral particles were used to infect the target cells with the addition of polybrene (8 µg/ml).

Quantitative real-time PCR

Total cellular RNA was isolated from PC3 and LNCaP cells by using TRIzol (Invitrogen) in accordance with the manufacturer’s instructions. RNA quality and quantity were measured using nanodrop ND-1000. Reverse transcription was performed using PrimeScript RT Reagent kit with gDNA Eraser (TaKaRa) and random primer. The following primer sequences were used in the real-time PCR: HPRT: forward, 5′-CCT GGC GTC GTG ATT AGT GAT-3′, reverse, 5′-AGA CGT TCA GTC CTG TCC ATAA-3′; DAB2: forward, 5′-GTA GAA ACA AGT GCA ACC AAT GG-3′, reverse, 5′-GCC TTT GAA CCT TGC TAA GAGA-3′. Real-time PCR was performed with SYBR premix Ex Taq (TaKaRa). Relative gene expression was calculated using the ΔΔCt method, with HPRT as internal control.

Western blot analysis

PC3 and LNCaP cells were collected and lysed using RIPA solution for total proteins. Proteins were separated by 10% SDS-PAGE, transferred onto a PVDF membrane, and then detected by an anti-DAB2 antibody. β-actin was used as internal control. Western blots were developed with Millipore Chemiluminescent HRP Substrate.

Cell migration assay

Scratch wound healing assays were performed to detect the cell migration ability. PC3 cells were transfected with scramble or DAB2 shRNAs. After 24 h, cells were inoculated into six-well plates following the same density. Cells were cultured for another 24 h via a 10 µl sterile pipette tip to make a straight scratch on adherent cells in each well, thereby stimulating a wound. After removing the suspended cells with PBS, adherent cells were cultured in a medium with low serum concentration (1%). Subsequently, the same location of cell migration was recorded by a camera at 0, 12, 24, and 36 h. Transwell chamber system was also used to detect the LNCaP cell migration ability.

Cell invasion assay

Transwell chamber invasion assays were performed to detect the cell invasion capability. PC3 cells were infected with lentiviruses carrying DAB2-shRNA or scramble shRNA, and LNCaP cells were transfected with MigR1-DAB2-GFP plasmid for 48 h. Up to 5 × 10⁴ cells from each group were seeded into the BioCoat Matrigel Invasion Chamber (BD Bioscience). About 0.5 ml of serum-free culture medium was added to the upper chamber. The lower chamber contained 20% FBS. Cells were allowed to invade for 24 h. Cells that penetrated through the Matrigel to the underside membrane surfaces were fixed and stained with 1% crystal violet.

Tumor formation assay

Tumor formation assay was performed as previously described [ 18]. Briefly, up to 5 × 10⁶ PC3 cells transfected with scramble and DAB2 shRNA were subcutaneously inoculated into SCID mice. Four weeks later, the mice were euthanized, and tumors were removed and fixed in formalin for several days. Subsequently, tumors were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological analysis. Up to 2 × 10⁶ PC3 cells transfected with scramble and DAB2 shRNA were also injected into the tail vein of the SCID mice to compare their effects on lung metastasis development. Five weeks later, the mice were euthanized, and lungs were removed and fixed in formalin for several days. Subsequently, lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological analysis. Mice were utilized in accordance with the animal care standards, and all protocols were approved by the Committee of Animal Use for Research at Shanghai Jiao Tong University School of Medicine (China).

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections (5 µm) were deparaffinized through routine techniques and incubated with 3% peroxide solution at room temperature for endogenous peroxidase ablation after target retrieval. Slides were blocked with 3% BSA solution and incubated with primary antibody against DAB2 at 4 °C overnight. Sections were then dropped with the anti-rabbit IgG-HRP solution and incubated for 1 h at room temperature. GTVision III immunohistochemistry detection kit (GeneTech) was used for the detection.

DNA methylation assay

DNA methylation assay was performed as previously reported [ 18]. CpG Island Searcher was used to find the DAB2 CpG island. DNA was extracted from LNCaP and PC3 cells by using a genomic DNA purification kit (Wizard, A1120) following the manufacturer’s protocol. EZ DNA Methylation™ Kit (Zymo Research, D5001) was used to create bisulfite reaction on 1 µg of genome DNA in accordance with the manufacturer’s protocol. DNA methylation primers were designed by MethPrimer software to amplify a 700 bp fragment and then cloned into pGEM-T Easy Vector (Promega). Ten clones of each group were sequenced for analysis.

Statistical analysis

Data were presented as means±SEM, and comparisons were conducted using t-test. A probability of 0.05 or less was considered statistically significant.

Results

DAB2 expression correlates with PCa clinical progression

LNCaP and PC3 cell lines, which were generated from PCa patients with low and high metastatic potentials, respectively, were selected to investigate the role of DAB2 in tumor progression and metastasis (Fig. S1). Real-time PCR and Western blot results showed that DAB2 expression in LNCaP cells was significantly lower than that in PC3 cells (Fig. 1A and 1B), which was also observed in the histochemical assay (Fig. 1C). This expression pattern suggested that DAB2 correlates with PCa clinical progression. DAB2 histochemical assays were also conducted on clinical samples from patients at different PCa stages for verification (Table S1). A significant increase in DAB2 expression along with PCa progression was revealed (Fig. 1D and Fig. S2), which was consistent with the results generated from PCa cell lines.

DAB2 is required for PCa cell invasion and migration capability

The development of the cell migratory and invasive properties is the most critical event in cancer progression and metastasis. DAB2 was transfected into LNCaP cells, and cell invasion and migration assays were performed using Transwell chamber system to determine the association between the migratory and invasive abilities of DAB2 and PCa cells. Exogenous DAB2 expression was confirmed by real-time PCR and Western blot analyses (Fig. 2A and 2B). The results revealed that enforced DAB2 overexpression enhanced both LNCaP cell invasion and migration (Fig. 2C and 2D).

shRNA assays were performed in PC3 cells to knockdown DAB2 and further verify the association between DAB2 and cell invasion/migration during PCa progression; the knockdown efficiency was confirmed by real-time PCR and Western blot (Fig. 3A and 3B). PC3 cell invasion/migration capability was then analyzed using Transwell chamber system and scratch wound healing assay. DAB2 knockdown by shRNA significantly inhibited PC3 cell invasion/migration (Fig. 3C‒3E). Enforced DAB2 overexpression can rescue the abnormal PC3 cell phenotype produced by DAB2 shRNA. Cell cycle, growth, and apoptosis analyses of PC3 cells transfected with scramble and DAB2 shRNA also revealed that DAB2 was dispensable for proliferation and apoptosis of PC3 cells (Fig. S3).

As described above, DAB2 was associated with PCa cell invasion and migration. The expression levels of some migration-related genes (including migration inhibitory factors RhoB, MMP2, and MMP9 and migration-activating factors RhoA, MMP10, and FSCN1) [ 19- 23] were then analyzed between LNCaP and PC3 cells by using real-time PCR for further verification. Gene expression was also detected in LNCaP cells transfected with DAB2 and PC3 cells transfected with DAB2 shRNA. Significant differences were observed among the comparable groups (Fig. 4A‒4C), indicating that DAB2 was involved in the regulation of some migration-related genes in PCa cells.

Histone 4 acetylation is crucial for activation of DAB2 transcription in PCa cells

Previous studies reported that DAB2 transcription was regulated by DNA methylation [ 24]. Therefore, DNA methylation status of DAB2 promoter was analyzed using bisulfite sequencing PCR. However, DAB2 differential expression between LNCaP and PC3 cells correlating with DNA methylation was not revealed (Fig. S4).

The DAB2 histone acetylation status in LNCaP and PC3 cells was analyzed because of the important role of histone acetylation in the regulation of gene transcriptional activation. First, LNCaP cells were treated with histone deacetylation inhibitor TSA, and PC3 cells were treated with histone acetylation inhibitor C646 to assess the correlation between DAB2 transcription and histone acetylation. Real-time PCR analysis revealed that DAB2 expression was significantly altered by TSA and C646 (Fig. 5A and 5B). ChIP-qPCR analysis of histone 4 acetylation enrichment on DAB2 locus in TSA-treated LNCaP cells and C646-treated PC3 cells was then performed. TSA treatment significantly increased the histone 4 acetylation enrichment level on DAB2 locus in LNCaP cells, and C646 treatment decreased the level of that in PC3 cells (Fig. 5C). Therefore, histone 4 acetylation is required to activate DAB2 transcription in PCa cells.

DAB2 is required for PCa cell tumorigenicity and metastasis in vivo

Although DAB2 can specifically regulate PCa cell invasion/migration in vitro, regulation of PCa cell tumorigenic potential by DAB2 in vivo remains unclear. Therefore, PC3 cells transfected with scramble and DAB2 shRNA were subcutaneously inoculated into the SCID mice to compare the teratoma formation potential. Both groups formed tumors in 4 weeks (Fig. 6A), but the tumors in DAB2 shRNA group were much smaller than those in the scramble shRNA group (Fig. 6B and 6C).

DAB2 and scramble shRNA group cells were also injected into the tail vein of mice to compare their effects on lung metastasis development. Mice injected with DAB2 shRNA-transfected PC3 cells were observed a significant decrease in lung weight and lung metastasis compared with those injected with scramble shRNA-transfected PC3 cells (Fig. 6D‒6F). Furthermore, the histopathological change in lung structure was observed under microscopic examination. DAB2 shRNA can rescue the lung damage caused by PC3 cell vein injection (Fig. 6G). Therefore, DAB2 is required for PCa cell tumorigenic capability and metastasis in vivo.

Discussion

DAB2 has always been regarded as a tumor suppressor gene, which is supposed to inhibit tumor metastasis because of its low expression in many kinds of tumor cells. However, in this study, an opposite DAB2 role during tumor progression was described. DAB2 expression level in LNCaP cells, which demonstrated low metastatic potential, was significantly lower than that in PC3 cells with high metastatic potential (Fig. 1A‒1C). DAB2 expression level also showed a correlation with the TNM malignant tumor classification in clinical PCa samples. A significant increase in DAB2 expression along with PCa progression was revealed through histochemical assays (Figs. 1D and S2), which raised the interesting prospect that DAB2 may be required for PCa progression and involved in multiple functions in tumor transformation and progression.

Cell invasion and migration are the most critical events in cancer progression and metastasis. The current in vitro experiments provided evidence that enforced DAB2 overexpression can enhance LNCaP cell invasion and migration, whereas DAB2 knockdown can inhibit these properties in PC3 cells (Figs. 2 and 3). To exclude the interference of other factors, cell cycle, growth, and apoptosis were analyzed, and no interference was observed (Fig. S3). Teratoma formation and lung metastasis assays further demonstrated that DAB2 is required for PCa cell tumorigenicity and metastasis in vivo (Fig. 6).

PCa progression can be partially blocked by targeting DAB2 inhibition; hence, the transcriptional regulatory mechanism of DAB2 expression must be determined. A previous study demonstrated that DAB2 can be induced by various stimuli, such as TGF-β and ATRA [ 25, 26]. DAB2 can also be regulated by DNA methylation [ 24], but the DAB2 differential expression between LNCaP and PC3 cells correlating with DNA methylation was not revealed in the present study (Fig. S4). Histone acetylation is another major epigenetic modification involved in the regulation of gene transcriptional activation, and the dynamic state of histone acetylation and deacetylation is charged by HAT and HDAC. In the present study, histone 4 acetylation appeared to be crucial for activation of DAB2 transcription in PCa cells for the first time. TSA, a histone deacetylation inhibitor, and C646, a HAT P300 inhibitor, can alter the DAB2 expression levels in LNCaP and PC3 cells, respectively (Fig. 5). Hence, the role of C646 in inhibiting PCa cell migration and invasion in vivo and in vitro must be evaluated, which may provide a new prospect for PCa treatment in the future.

Although the current results elucidated an important role of DAB2 in PCa progression, the underlying molecular mechanisms remains unclear. The next question that should be addressed is the precise mechanism of how DAB2 regulates PCa cell migration and invasion during tumor progression. DAB2 can modulate tumor progression by promoting EMT-dependent metastasis [ 25], but further evidence is still needed for support, especially in vivo. DAB2 is also known as an important member of clathrin-coated pits and plays some roles in endocytosis [ 27]. A quantitative proteomics approach was developed by one group to confirm whether DAB2 can influence the integrin signaling cascade to control cell migration by regulating the endocytosis of integrin β1 [ 15]. Furthermore, DAB2 is a mitogen-responsive phosphoprotein, it can interact with Grb2 and modulate growth factors/RAS pathway [ 28]. Another study clarifies that DAB2 can directly stimulate JNK pathway [ 29]. MAPK signaling pathway also has important functions in the regulation of cell migration and invasion [ 30]. As adapter protein, DAB2 can interact with SMAD2/3 and DVL-3, indicating that DAB2 is required for the activation of TGF-β and Wnt pathway [ 31]. Nevertheless, regrettably, it still remains unclear which signal is actually involved in our study, and therefore, it will be a major direction for further research.

In conclusion, DAB2 is required for PCa cell invasion and migration capability in vivo and in vitro. Furthermore, DAB2 differential expression between early- and late-stage PCa cells was partly regulated by histone 4 acetylation. Therefore, DAB2 may play an important role in PCa progression and metastasis.

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