Chronic NaAsO2 exposure promotes migration and invasion of prostate cancer cells by Akt/GSK-3β/β-catenin/TCF4 axis-mediated epithelial-mesenchymal transition

doi:10.21203/rs.3.rs-3796580/v1

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Chronic NaAsO₂ exposure promotes migration and invasion of prostate cancer cells by Akt/GSK-3β/β-catenin/TCF4 axis-mediated epithelial-mesenchymal transition

https://doi.org/10.21203/rs.3.rs-3796580/v1

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Inorganic arsenic is a Class I human Carcinogen. However, the role of chronic inorganic arsenic exposure on prostate cancer metastasis still unclear. This study aimed to investigate the effect and mechanism of chronic NaAsO₂ exposure on migration and invasion of prostate cancer cells. DU145 and PC-3 cells were exposed to NaAsO₂ (2 µmol/L) for 25 generations. Wound healing and Transwell assays showed that chronic NaAsO₂ exposure promoted migration and invasion of DU145 and PC-3 cells. In addition, chronic NaAsO₂ exposure induced epithelial-mesenchymal transition (EMT) of DU145 cells by promoting β-catenin/TCF4 transcriptional activity. Mechanically, NaAsO₂ promoted GSK-3β inactivation in the "disruption complex" through Akt mediated phosphorylation at serine 9, and then inhibited phosphorylation and ubiquitination degradation of β-catenin, leading to β-catenin nuclear translocation. Ly204002, a selective phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, suppressed the β-catenin/TCF4 complex activation and EMT through blocking the Akt-mediated GSK-3β inactivation in the "disruption complex" in chronic NaAsO₂ exposed DU145 and PC-3 cells. Moreover, Ly204002 alleviated chronic NaAsO₂-induced migration and invasion in DU145 and PC-3 cells. These findings provide evidence that chronic arsenic exposure promoted migration and invasion of prostate cancer cells through inducing EMT driven by AKT/GSK-3β/β-catenin/TCF4 signaling axis. Akt is expected to be a potential therapeutic target for chronic arsenic exposure-mediated prostate cancer metastasis.

Prostate cancer

Chronic NaAsO2 exposure

Invasion

β-catenin/TCF4

Epithelial-mesenchymal transition

Arsenic is a naturally occurring metalloid and a Class I human Carcinogen (Chen et al. 2019; Li and Chen 2016). Usually, arsenic exists in the environment in its inorganic form of trivalent and pentavalent arsenate (Li and Chen 2016). The general population is usually chronic exposed to arsenic by intaking contaminated water, foods and air (Li and Chen 2016; Mao et al. 2020.). It is estimated that approximately 200 million people around the world are exposed to unsafe levels of arsenic (Podgorski and Berg 2020). Chronic arsenic exposure has been correlated with many types of cancer (e.g., lung, liver, bladder, kidneys, skin, prostate), as well as non-carcinogenic diseases such as skin lesions, cardiovascular disease, reproductive defects, neurological injuries, and diabetes mellitus (Chen et al. 2019; Benbrahim-Tallaa and Waalkes 2008; Speer et al. 2023). Several studies indicated that long-term exposure to arsenic increases the risk of prostate cancer morbidity and mortality (Garcia-Esquinas et al. 2013; Bulka et al. 2016; Roh et al. 2017). Our previous research found that chronic arsenic exposure enhanced stemness and chemoresistance of prostate cancer cells (Zhang et al. 2021). However, the effects of chronic arsenic exposure on prostate cancer metastasis and its mechanisms still unclear.

Prostate cancer is one of the most common malignant tumors in male urinary system. With an estimated almost 1.4 million new cases and 375,000 deaths worldwide in 2020 (Sung et al. 2021). Metastasis is the leading cause of prostate cancer-related death, and the 5-year survival rate for patients with metastatic prostate cancer is only 31% (Swami et al. 2020). The current opinion is that epithelial-mesenchymal transformation (EMT) is one of the key mechanisms inducing tumor cell metastasis. EMT is a cellular process during which epithelial cells acquire mesenchymal phenotypes and behavior following the downregulation of epithelial features (Pastushenko and Blanpain 2019; Yang et al. 2020). Tumor cells undergoing EMT have stronger migratory and invasive capabilities, which facilitates distant metastasis of tumor cells from the primary site. Several important pathways, such as TGF-β/Smad2/3, β-catenin/TCF4, PI3K/AKT and IL-6/STAT3, driving EMT of tumor cells (Zhang et al. 2016; Yang et al. 2017; Yu er al. 2018; Xu et al. 2020; Ma et al. 2023).

This study aims to explore the effect and mechanism of EMT in metastasis of prostate cancer cells mediated by chronic arsenic in vitro. Our results showed that chronic arsenic exposure enhanced migration and invasion abilities of prostate cancer cells. In addition, chronic arsenic exposure induced EMT by activating AKT/GSK-3β/β-catenin/TCF4 axis. Moreover, PI3K/AKT inhibitor significantly suppressed chronic arsenic-mediated migration and invasion of prostate cancer cells by inhibiting GSK-3β/β-catenin/TCF4 axis-driven EMT. We provide novel evidence that chronic arsenic exposure aggravates migration and invasion of prostate cancer cells through inducing EMT driven by AKT/GSK-3β/β-catenin/TCF4 axis.

Chemicals and reagents

Sodium arsenite (NaAsO₂) was purchased from Sigma Chemical Co. (St. Louis, MO). CCK-8 kits were from Biosharp (Shanghai, China). LY294002 was from LC Laboratories (Woburn, MA). Antibodies against E-cadherin, N-cadherin, Vimentin, Snail, Twist1, Axin1, β-catenin, phosphor-β-catenin (p-β-catenin), Akt, phosphor-Akt (Ser473), GSK3β, phosphor-GSK3β (Ser9), MMP-2, MMP-9, β-actin, Ubiquitin and Lamin A/C were purchased from Cell Signaling Technology (Beverley, MA). Zeb1 and TCF4 antibodies were from Abcam (Cambridge, MA). Immunoglobulin secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). TRI reagent was from Molecular Research Center, Inc (Cincinnati, Ohio). RNase-free DNase and Avian Myeloma Virus reverse transcriptase (AMV reverse transcriptase) were from Promega (Madison, WI). Chemiluminescence (ECL) detection kit was from Pierce Biotechnology (Rockford, IL). All the other reagents were from Sigma Chemical Co. (St. Louis, MO) or as indicated in the specified methods.

Cell culture and treatments

PC-3 and DU145, two human prostate cancer cell lines, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). PC-3 cells were grown in F-12 HAM’S Medium (HyClone) supplemented with 100 U/mL of penicillin, 100 µg/mL streptomycin and 10% FBS (Gibco) at 5% CO₂, 37°C. DU145 cells were cultured in MEM medium (Invitrogen) containing penicillin (100 U/mL), streptomycin (100 µg/mL), GlutaMAX (1%, Gibco), sodium pyruvate (1 mM, Gibco), Non-essential Amino Acids (0.1 mM) and 5% FBS (Gibco) at 5% CO₂, 37°C. Chronic NaAsO₂-exposed PC-3 and DU145 cells were constructed by co-cultured with NaAsO₂ (2 µmol/L) for 25 generations. At the same time, parental PC-3 and DU145 cells with the same passages were constructed. To investigate the antagonistic effects of LY294002 on NaAsO₂-mediated migration, invasion and EMT of prostate cancer cells. Chronic NaAsO₂-exposed DU145 and PC-3 cells were incubated with LY294002 (50 µM) for 5 generations. Cells were harvested for wound healing, Transwell, Western blotting, RT-PCR, Co-immunoprecipitation (Co-IP), and Immunofluorescence (IF) assays.

Wound healing migration assay

Wound healing assay was carried out as described by others with minor modifications (Xing et al. 2020). Briefly, PC-3 and DU145 cells (5.0 ×10⁵ cells/well) were cultured in six-well plates until 100% confluency. The confluent monolayer cells were carefully scratched using a 200-µL tip and washed twice with PBS. Cells were imaged at low magnifications for time intervals of 0 and 24 hours under a light microscope. The wounded area was calculated according to the formula: (mean wounded breadth - mean remained breadth) /mean wounded breadth×100 (%).

Transwell assays

Migration and invasion activities of PC-3 and DU145 cells were assessed using 8-µm Transwell filters (Costar Corning) as described previously with minor modifications (Zhang et al., 2023). For migration assays, 4 × 10⁴ cells in 200 µL serum-free medium were seeded in the upper compartment. After 12 h incubation, add 60 µL FBS (10%) to the lower chamber as a chemoattractant and then incubate for 12 hours. For invasion assays, 1.0×10⁵ cells in 200 µL complete medium were seeded in the upper compartment, precoated with 50 µL Matrigel solution (BD Biosciences, San Jose, CA). After incubation for 36 h, the lower compartments were added to 60 µL FBS as a chemoattractant and incubated for 12 h. Non-migratory cells on the upper side of the membrane were wiped carefully with a wet cotton swab. The membranes were fixed with methanol for 20 min and stained with 0.1% crystal violet solution for 20 min. The numbers of cells that migrated to the bottom of the filter were enumerated by detecting the absorbance of decolorization solution of 30% acetic acid at 570 nm wavelength. These experiments were performed three times independently.

Protein extraction and Western blotting

Total, cytoplasmic and nuclear proteins were extracted according to previous method (Xing et al. 2020). Same amount (10–30 µg) of protein was separated electrophoretically by SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated for 2 h with following primary antibodies: E-cadherin (1:2,000), N-cadherin (1:1,000), Vimentin (1:2,000), ZEB1 (1:1,000), Snail (1:1,000), Twist (1:1,000), Axin1 (1:2000), β-catenin (1:2,000), p-β-catenin (1:1,000), p-Akt^Ser473 (1:1,000), Akt (1:2,000), p-GSK3β^Ser9 (1:2,000), GSK3β (1:2,000), MMP-2 (1:1,000), MMP-9 (1:1,000), TCF4 (1:1,000) and Ubiquitin (1:1,000). β-actin (1:10,000), α-tublin (1:5,000) and Lamin A/C (1:5,000) or Lamin B1 (1:3,000) were used as loading controls for total, cytoplasmic and nuclear proteins, respectively. After incubated with goat anti-rabbit IgG or goat anti-mouse antibody for 1 ~ 2 hours, fluorescence signals were detected using an ECL detection kit according to the manufacturer’s instruction.

Co-immunoprecipitation (Co-IP) assay

Co-immunoprecipitation experiment was carried out as described previously (Xing et al., 2020). Briefly, cytoplasmic and nuclear proteins (800 µg) were precleared with protein A/G-agarose (Santa Cruz) and then incubated with anti-Axin and anti-TCF4 antibodies at 4˚C overnight, respectively. The precipitates were washed with cold non-denaturing lysis buffer before collecting the agarose-trapped immune complex. The immune complex was separated by boiling and used for subsequent Western blotting analysis.

Isolation of total RNA and real-time PCR

Total RNA was extracted from DU145 cells using TRIzol reagent according to manufacturer’s protocol. RNA (2.0 µg) was then reverser-transcribed into cDNA using AMV kit (Promega). Real-time PCR was performed as described previously (Xing et al., 2020). Briefly, cDNA was amplification with a LightCycler® 480 SYBR Green I kit (Roche Diagnostics GmbH, Mannheim, Germany) using specific primers as listed in Table 1. The amplification reactions were carried out on a LightCycler® 480 Instrument (Roche Diagnostics GmbH) with an initial hold step (95°C for 5 minutes) and 50 cycles of a three-step PCR (95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds). The comparative CT-method was used to determine the relative amount of target genes, normalized to an endogenous reference (18S), and relative to a calibrator using the LightCycler 480 software (Roche, version 1.5.0).

Table 1

Oligonucleotide sequence of primers for real-time RT-PCR
Gene	Sequences
18S	Forward: 5’-CGGCTACCACATCCAAGGAA-3’ Reverse: 5’-GCTGGAATTACCGCGGCT-3’
Axin-2	Forward: 5’-AATAAGGCGAAGATCAACATGGC-3’
Axin-2	Reverse: 5’-TTTGTTACCAATGTCCCCAAGAG-3’
Cyclin D1	Forward: 5’-GCTGCGAAGTGGAAACCATC-3’
Cyclin D1	Reverse: 5’- CCTCCTTCTGCACACATTTGAA-3’
c-Myc	Forward: 5’-GGCTCCTGGCAAAAGGTCA-3’
c-Myc	Reverse: 5’-CTGCGTAGTTGTGCTGATGT-3’
Twist1	Forward: 5’-GTCCGCAGTCTTACGAGGAG-3’
Twist1	Reverse: 5’-GCTTGAGGGTCTGAATCTTGCT-3’
Zeb1	Forward: 5’-GATGATGAATGCGAGTCAGATGC-3’
Zeb1	Reverse: 5’-ACAGCAGTGTCTTGTTGTTGT-3’
Snail	Forward: 5’-TCGGAAGCCTAACTACAGCGA-3’
Snail	Reverse: 5’-AGATGAGCATTGGCAGCGAG-3’
E-cadherin	Forward: 5’-CGAGAGCTACACGTTCACGG-3’
E-cadherin	Reverse: 5’-GGGTGTCGAGGGAAAAATAGG-3’
N-cadherin	Forward: 5’-AGCCAACCTTAACTGAGGAGT-3’
N-cadherin	Reverse: 5’-GGCAAGTTGATTGGAGGGATG-3’
Vimentin	Forward: 5’-GACGCCATCAACACCGAGTT-3’
Vimentin	Reverse: 5’-CTTTGTCGTTGGTTAGCTGGT-3’

Immunofluorescence (IF) assays

Immunofluorescence (IF) assays was performed according to a previous report (Zhang et al. 2023). Cells were cultured in 24-well cell slides and fixed with methanol. After blocked with BSA, anti-β-catenin primary antibody (1:200) was used to incubated cells at 4˚C overnight. After PBS washing, cells were incubated with a species-specific FITC-conjugated secondary antibody (1:500; Invitrogen, Carlsbad, CA, USA). Nuclei were stained with DAPI. Photographs were taken with confocal microscope (LSM880, Zeiss).

Statistical analysis

SPSS 13.0 statistics software was used for data analysis. All data were expressed as mean ± SEM. The differences between two groups were analyzed using Student’s t-test. ANOVA and the Student-Newmann-Keuls post hoc tests were used to determine differences among more than two groups. All statistical tests were two-sided using an alpha level of 0.05.

Chronic NaAsO₂ exposure promotes migration and invasion of prostate cancer cells

Effects of chronic NaAsO₂ exposure on migration and invasion of prostate cancer cells were analyzed. Wound healing assays showed that the percentage of wound closing were significantly increased in chronic NaAsO₂-treated DU145 and PC-3 cells (Fig. 1a). Transwell assays showed that cell migration (Fig. 1b) and invasion (Fig. 1c) were enhanced in NaAsO₂-treated DU145 and PC-3 cells. In addition, protein levels of MMP2 and MMP9 were elevated in NaAsO₂-treated DU145 cells (Fig. 1d).

Chronic NaAsO₂ exposure induces epithelial-mesenchymal transition of prostate cancer cells

The effect of chronic NaAsO₂ exposure on epithelial-mesenchymal transition was analyzed in prostate cancer cells. As shown in Fig. 2a and b, mRNA and protein levels of E-cadherin, an epithelial marker, were decreased in chronic NaAsO₂-treated DU145 cells. By contrast, N-cadherin and vimentin, two mesenchymal markers, were upregulated both mRNA and protein in chronic NaAsO₂-treated DU145 cells. In addition, mRNA levels of Zeb1, Twist1 and Snail, three key EMT-transcription factors, were increased in chronic NaAsO₂-treated DU145 cells (Fig. 2c). Moreover, nuclear Zeb1, Twist1 and Snail levels were elevated in chronic NaAsO₂-treated DU145 cells (Fig. 2d).

Chronic NaAsO₂ exposure promotes β-catenin/TCR4 transcription by activating Akt/GSK-3β signal in prostate cancer cells

The effect of chronic NaAsO₂ exposure on β-catenin/TCR4 signal was analyzed in prostate cancer cells. Immunofluorescence assays showed that fluorescence intensities of nuclear β-catenin were enhanced in NaAsO₂-treated DU145 and PC-3 cells (Fig. 3a). In addition, nuclear β-catenin level was significantly increased in NaAsO₂-treated DU145 cells (Fig. 3b). Co-IP assay showed that the interaction between β-catenin and TCF4 was enhanced in NaAsO₂-treated DU145 cells (Fig. 3c). As a result, Axin-2, Cyclin D1 and c-Myc mRNAs, three downstream target genes of β-catenin/TCF4 complex, were up-regulated in NaAsO₂-treated DU145 cells (Fig. 3d). The effect of chronic NaAsO₂ exposure on Akt/GSK-3β pathway was investigated in DU145 cells. As shown in Fig. 3e, Akt phosphorylation on serine 473 (Akt^Ser473) was elevated in NaAsO₂-treated DU145 cells. Moreover, total GSK-3β phosphorylation on serine 9 (GSK3β^Ser9) was upregulated in NaAsO₂-treated DU145 cells (Fig. 3e). Next, we investigated the effect of chronic NaAsO₂ exposure on GSK-3β phosphorylation on serine 9 in the “destruction complex” by Co-immunoprecipitation. As shown in Fig. 3f, p-GSK3β^Ser9 in the “destruction complex” was upregulated in NaAsO₂-treated DU145 cells. As expected, phosphorylation level of cytoplasmic β-catenin was decreased in NaAsO₂-treated DU145 cells (Fig. 3g). Moreover, ubiquitination of cytoplasm β-catenin was attenuated in NaAsO₂-treated DU145 cells (Fig. 3h).

Ly294002 inhibits NaAsO₂-induced β-catenin/TCR4 activation of DU145 cells

The effects of Ly294002, a selective PI3K/Akt inhibitor, on β-catenin/TCR4 signal were analyzed in NaAsO₂-treated prostate cancer cells. As shown in Fig. 4a and b, Ly294002 significant down-regulated phosphorylation levels of Akt^Ser473 and GSK3β^Ser9 both in NaAsO₂-treated and -untreated DU145 cells. In addition, Ly294002 downregulated nuclear β-catenin level both in NaAsO₂-treated and -untreated DU145 cells (Fig. 4c and d). Moreover, Ly294002 upregulated phosphorylation level of cytoplasmic β-catenin both in NaAsO₂-treated and -untreated DU145 cells (Fig. 4e and f). Co-IP assay showed that Ly294002 promoted ubiquitinated degradation of cytoplasm β-catenin both in NaAsO₂-treated and -untreated DU145 cells (Fig. 4g and h). Correspondingly, Ly294002 downregulated downstream target genes expression of β-catenin/TCF4 signal both in NaAsO₂-treated and -untreated DU145 cells (Fig. 4i).

Ly294002 suppresses NaAsO₂-induced EMT of DU145 cells

The inhibitory effect of PI3K/Akt inhibitor on EMT was then analyzed in NaAsO₂-treated prostate cancer cells. As shown in Fig. 5a and b, Ly294002 upregulated mRNA and protein levels of E-cadherin both in NaAsO₂-treated and -untreated DU145 cells. By contrast, Ly294002 reduced mRNA and protein levels of N-cadherin and vimentin both in NaAsO₂-treated and -untreated DU145 cells (Fig. 5a and b). In addition, Ly294002 decreased mRNA and nuclear protein levels of Zeb1, Twist1 and Snail both in NaAsO₂-treated and -untreated DU145 cells (Fig. 2c and d).

Ly294002 alleviates NaAsO₂-induced migration and invasion of DU145 and PC-3 cells

Antagonistic effects of PI3K/Akt inhibitor on chronic NaAsO₂ exposure-mediated migration and invasion were analyzed in prostate cancer cells. Wound healing assays showed that Ly294002 downregulated wound closing percentage both in chronic NaAsO₂ treated DU145 and PC-3 cells (Fig. 6a). Transwell assays showed that Ly294002 inhibited migration (Fig. 6b). and invasion (Fig. 6c) both in chronic NaAsO₂-treated and -untreated DU145 and PC-3 cells.

The major findings of this study are as follow. Firstly, chronic NaAsO₂ exposure enhanced migration and invasion of prostate cancer cells. Secondly, chronic NaAsO₂ exposure induced EMT by promoting β-catenin/TCF4 complex transcriptional activity due to Akt-mediated inactivation of GSK-3β in the “destruction complex” and nuclear translocation of cytoplasmic β-catenin. Thirdly, PI3K/Akt inhibitor suppressed the β-catenin/TCF4 complex activation through blocking the phosphorylation of GSK3β^Ser9 in the β-catenin “destruction complex”. Fourthly, PI3K/Akt inhibitor alleviated chronic NaAsO₂ exposure-mediated migration and invasion by suppressing EMT of prostate cancer cells. These findings provide evidence that chronic arsenic exposure promoted migration and invasion of prostate cancer cells through inducing EMT driven by Akt/GSK-3β/β-catenin/TCF4 axis.

The carcinogenic roles of inorganic arsenic have been widely investigated (Saintilnord and Fondufe-Mittendorf 2021; Wadgaonkar and Chen 2021). The current study investigated the effects of chronic arsenic on migration and invasion of prostate cancer cells and its underlying mechanism. Our results showed that chronic NaAsO₂ exposure enhanced migration and invasion abilities of DU145 and PC-3 cells. Accumulating evidence suggests that EMT is the initiating event that drives tumor metastasis (Yang et al. 2020; Bakir et al. 2020). EMT is a cellular process during which epithelial cells acquire mesenchymal phenotypes and behavior following the downregulation of epithelial features (Yang et al. 2020). Cells that undergo EMT tend to be more migratory and aggressive. In the present study, our results showed that mRNA and protein levels of E-cadherin, an epithelial marker, were significantly down-regulated in chronic NaAsO₂-treated DU145 cells. By contrast, N-cadherin and vimentin, two mesenchymal markers, were up-regulated in NaAsO₂-treated DU145 cells. In addition, EMT-transcription factors Zeb1, Twist1 and Snail mRNAs and nuclear protein levels were elevated in chronic NaAsO₂-treated DU145 cells. These results suggest that chronic arsenic exposure enhances prostate cancer cell migration and invasion by inducing EMT.

Previous studies have demonstrated that the Akt/GSK-3β/β-catenin/TCF4 axis is one of the key signals driving EMT in tumor cells (Pan et al. 2019; Song et al. 2020). As the core kinase of the β-catenin destruction complex, GSK-3β first induces phosphorylation of β-catenin, which facilitates its ubiquitinated degradation (Stamos and Weis 2013; Ranes et al. 2021). GSK-3β inactivation in the “destruction complex” leads to the cytoplasmic β-catenin accumulation which in turn causes the transnucleation of β-catenin and triggers target genes transcription by binding to TCF/LEF (Schaefer and Peifer 2019). Some of these target genes are EMT transcription factors (e.g. Zeb1 and Twist) and Matrix metalloproteinase (e.g. MMP2 and MMP7) (Sánchez-Tilló et al. 2011; Heuberger and Birchmeier 2010). Here, our results showed that chronic NaAsO₂ exposure induced Akt phosphorylation activation, which then resulted in inactivation of cytoplasm GSK-3β via phosphorylation at serine 9 (Liu et al. 2014; Beurel et al. 2015). Considering that only GSK-3β present in the “destruction complex”, rather than free GSK-3β in the cytoplasm, can catalyze the phosphorylation of β-catenin. We further investigated the effect of chronic NaAsO₂ exposure-mediated Akt activation on GSK-3β activity in the “destruction complex”. Co-IP assay showed that NaAsO₂ significantly elevated the phosphorylation of GSK-3β at serine 9 in the “destruction complex”, indicating that chronic NaAsO₂ exposure leads to GSK-3β inactivation in the “destruction complex”. In addition, chronic NaAsO₂ exposure reduced cytoplasmic β-catenin phosphorylation and ubiquitination, leading to its nucleus accumulation in DU145 cells. Subsequent results showed that the interaction between β-catenin and TCF4 was enhanced in NaAsO₂-treated DU145 cells. As expected, the levels of Axin2, Cyclin D1 and c-Myc mRNAs, several downstream target genes of β-catenin/TCF4 complex, were up-regulated in NaAsO₂-treated DU145 cells. These results suggest that chronic NaAsO₂ exposure may promote EMT through Akt-mediated GSK3β/β-catenin/TCF4 signaling in prostate cancer cells.

Next, we investigated the antagonistic effects of PI3K/Akt inhibitors on GSK-3β/β-catenin/TCF4 signaling and EMT in chronic NaAsO2-treated prostate cancer cells. Our results showed that Ly294002, a selective PI3K/Akt inhibitor, inhibited phosphorylation of Akt at serine 473 and GSK-3β at serine 9 both NaAsO₂-treted or untreated DU145 cells. In addition, Ly294002 increased cytoplasmic β-catenin phosphorylation and subsequent ubiquitination degradation, thereby inhibiting β-catenin nuclear accumulation in NaAsO₂-treated and -untreated DU145 cells. As expected, Ly294002 suppressed the expression of downstream target genes of β-catenin/TCF4 complex, indicating that Akt inhibitor antagonized GSK-3β/β-catenin/TCF4 signal transduction in chronic arsenic-exposed prostate cancer cells. Further results showed that Ly294002 inhibited EMT in chronic NaAsO₂-treated and -untreated DU145 cells. Moreover, Ly294002 downregulated migration and invasion in chronic NaAsO₂-treated and -untreated DU145 cells. These results suggest that Akt inhibitor suppress migration and invasion by antagonizing EMT induced by the AKT/GSK-3β/-β-catenin/TCF4 axis in arsenic-exposed prostate cancer cell.

In summary, the present study investigated the effects of chronic arsenic exposure on prostate cancer cell migration and invasion and its molecular mechanisms. Our results found that chronic NaAsO₂ exposure promoted migration and invasion of prostate cancer cells. Mechanistically, chronic NaAsO₂ exposure induced EMT through activating AKT/GSK-3β/-β-catenin/TCF4 signal transduction in chronic arsenic-exposed prostate cancer cells. Akt inhibitor suppressed migration and invasion by antagonizing EMT induced by the Akt/GSK-3β/-β-catenin/TCF4 axis in arsenic-exposed prostate cancer cell. Akt is expected to be a potential therapeutic target for chronic arsenic exposure-mediated prostate cancer metastasis.

AKT

protein kinase B

Co-IP

Co-immunoprecipitation

EMT

epithelial-mesenchymal transition

GSK-3β

glycogen synthase kinase-3β

immunofluorescence

MMP

matrix metalloproteinases

PCR

polymerase chain reaction

PI3K

phosphatidylinositol 3 kinase

Snail

snail family transcriptional repressor 1

TCF4

T cell-specific factor

TGF-β

transforming growth factor-β

Twist1

twist family bHLH transcription factor 1

STAT3

signal transducer and activator of transcription 3

Zeb1

zinc finger E-box binding homeobox 1

Author contributions

Conception and design: Z-H Z and D-D X; Experiment performance and data acquisition: Z-H Z, H-X Y, M-D L and K Y; Analysis and interpretation of data: H-X Y, F-W N, S-Y F, X L and Y-H C; Funding acquisition: Z-H Z and D-D X; Roles/Writing-original draft: Z-H Z; Writing, review, and/or revision of the manuscript: Z-H Z and D-D X.

Acknowledgements

We thank Prof. Zheng-Mei Lv (from Department of Histology and Embryology, Anhui Medical University, Hefei, 230032, China) for her assistance.

Ethics approval

Not applicable.

Funding

This work was supported by the Anhui Medical University Translational Medicine Program (2021zhyx-C58), Anhui Provincial Natural Science Foundation (2108085MH261), and University Natural Science Research Project of Anhui Province (KJ2021A0318).

Conflict of interest

The authors declare no conflict of interest.

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Chronic NaAsO₂ exposure promotes migration and invasion of prostate cancer cells by Akt/GSK-3β/β-catenin/TCF4 axis-mediated epithelial-mesenchymal transition

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Chemicals and reagents

Cell culture and treatments

Wound healing migration assay

Transwell assays

Protein extraction and Western blotting

Co-immunoprecipitation (Co-IP) assay

Isolation of total RNA and real-time PCR

Immunofluorescence (IF) assays

Statistical analysis

Results

Chronic NaAsO₂ exposure promotes migration and invasion of prostate cancer cells

Chronic NaAsO₂ exposure induces epithelial-mesenchymal transition of prostate cancer cells

Chronic NaAsO₂ exposure promotes β-catenin/TCR4 transcription by activating Akt/GSK-3β signal in prostate cancer cells

Ly294002 inhibits NaAsO₂-induced β-catenin/TCR4 activation of DU145 cells

Ly294002 suppresses NaAsO₂-induced EMT of DU145 cells

Ly294002 alleviates NaAsO₂-induced migration and invasion of DU145 and PC-3 cells

Discussion

Abbreviations

Declarations

References

Additional Declarations

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Version 1

Chronic NaAsO2 exposure promotes migration and invasion of prostate cancer cells by Akt/GSK-3β/β-catenin/TCF4 axis-mediated epithelial-mesenchymal transition

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Chemicals and reagents

Cell culture and treatments

Wound healing migration assay

Transwell assays

Protein extraction and Western blotting

Co-immunoprecipitation (Co-IP) assay

Isolation of total RNA and real-time PCR

Immunofluorescence (IF) assays

Statistical analysis

Results