PTP4A1 promotes intrahepatic cholangiocarcinoma development and progression by interacting with PTEN and activating the PI3K/AKT/GSKα axis

Introduction

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive and malignant liver cancer that accounts for 10–20% of primary liver cancers and is the second most common type after hepatocellular carcinoma (HCC) (1,2). The incidence of ICC has been increasing globally, with an average annual growth rate of 4.4% over the past decade (3). Unfortunately, most patients are diagnosed at advanced stages because of the asymptomatic nature of early ICC, leading to limited treatment options and poor clinical outcomes (4). The 5-year survival rate remains dismal, at ~25-40% even after curative resection, with a high recurrence rate (5). Surgical resection is currently the only potentially curative treatment for ICC, but it is suitable for only a minority of patients. The molecular mechanisms underlying ICC growth and metastasis are not well understood, which hinders the development of new therapies. Factors such as chronic inflammation, genetic mutations in genes such as isocitrate dehydrogenase (NADP+ IDH 1/2), epidermal growth factor receptors, fibroblast growth factor receptors and ROS proto-oncogene 1, as well as aberrant signaling pathways, contribute to ICC development (6–8). However, the clear mechanisms of ICC have not been fully elucidated, and there is an urgent need to investigate the molecular pathogenesis of ICC to identify new therapeutic targets and approaches.

PTP4A1, also known as protein tyrosine phosphatase 4A1, is an enzyme that belongs to the protein tyrosine phosphatase (PTP) family. It plays significant roles in various cellular processes, including cell proliferation, differentiation and migration (9–11). PTP4A1 is involved in the dephosphorylation of tyrosine residues on proteins, which can influence the PI3K/AKT, ERK and transforming growth factor beta 1 (TGFβ) signaling pathways (12–15). PTP4A1 has also been implicated in the development and progression of several types of cancer, including non-small cell lung cancer (NSCLC) (9), HCC (16), cervical cancer (13) and colon cancer (17), where it may promote cell proliferation, survival and metastasis. High levels of PTP4A1 expression are associated with poor prognosis in various types of cancer (9,18,19), suggesting its potential as a therapeutic target and biomarker. A previous study by the authors revealed that the lncRNA NEAT1 promoted cell proliferation, migration and invasion via the miR-186-5p/PTP4A1 axis in cholangiocarcinoma (20). However, the function and mechanism of PTP4A1 in ICC remain largely unclear.

In the present study, the potential role and underlying molecular mechanism of PTP4A1 in ICC were explored. Our results revealed that PTP4A1 was upregulated in ICC and associated with lymph node metastasis, vascular invasion, tumor node metastasis (TNM) stage and differentiation of ICC. Furthermore, it was demonstrated that PTP4A1 promoted the proliferation, metastasis and invasion of ICC. In terms of mechanism, PTP4A1 interacts with PTEN, decreases the phosphorylation of PTEN and promotes the activation of the PI3K/AKT pathway. Therefore, our results suggest that PTP4A1 is a promising therapeutic target for ICC.

Materials and methods

Patients and specimens

ICC tissues and matched adjacent non-tumor tissues were obtained from 60 patients (37 men and 23 women; median age, 64 years; age range, 41–83 years) who underwent radical resection for ICC at the Hunan Provincial People's Hospital (Changsha, China) between January 2021 and January 2023. The diagnoses were pathologically confirmed by two independent pathologists. None of the patients received any anticancer therapy prior to surgery. A total of 40 pairs of fresh ICC specimens and matched adjacent non-tumor tissues were stored in liquid nitrogen immediately after resection and subsequently stored at −80°C for reverse transcription-quantitative PCR (RT-qPCR) and WB. A total of 20 pairs of ICC specimens and matched adjacent non-tumor tissues were fixed in 4% paraformaldehyde (PFA) at room temperature for 12–24 h and subsequently subjected to paraffin embedding and immunohistochemical (IHC) analysis. All experiments in our research were approved (approval no. 2023–151) by the Ethics Committee of the Hunan Provincial People's Hospital (Changsha, China) and carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.

Sample size selection and power calculation

In more than 75% of normal lung tissue samples, the percentage of PTP4A1-positive cells among the total cells is <50%, which is considered as low expression of PTP4A1 (9). In the present study, it was assumed that the probability of PTP4A1 overexpression (OE) in ICC adjacent tissues (P1) was 0.3, and that in the ICC tissue (P2) was 0.7. Using the T-test method, with a significance level (ɑ) set at 0.05 and a statistical power (β) set at 0.8, the calculated sample size based on the sample size formula {n=(Zɑ/2+Zβ)2 × [p1(1-p1) + p2 (1-p2)]/(p1-p2)2]} is 21. Using G*Power, it was found that a sample size of 21 per group achieves a power of ~0.8 (80%). In the present study, IHC results are subjective to some extent and may be affected by technical variability. Therefore, a relatively small sample size is needed to balance resource consumption and the reliability of the results. A sample size of 20 pairs was selected to avoid excessive experimental costs and workload. Western blotting (WB) and RT-qPCR are quantitative detection techniques that can accurately measure the expression levels of PTP4A1, with high sensitivity and specificity. A total of 10 pairs of ICC tumor tissues and adjacent tissues were analyzed by to determine the expression of PTP4A1. For RT-qPCR assay, a sample size of 40 pairs can provide sufficient statistical power to identify potential association between PTP4A1 expression and ICC clinical features.

Cell culture and transfection

The ICC cell lines HCCC-9810 and RBE were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a 5% CO2, 37°C incubator. Lentiviruses containing specific short hairpin RNA (shRNA) targeting PTP4A1 (sh-PTP4A1: ATCCAACCAATGCGACCTT) or shRNA negative control (sh-NC: TTCTCCGAACGTGTCACGT) were purchased from Shanghai GenePharma Co., Ltd. The PTP4A1 coding sequence was synthesized, cloned and inserted into a lentiviral GV492 vector. The empty GV492 vector was considered an empty vector (EV). A total of 5×104 HCCC-9810 and RBE cells were seeded into 6-well plates for 24 h. When the cells confluence was 30%, they were infected with the appropriate lentiviruses. The optimal infection conditions were 10 µl lentivirus (108 TU/ml) + 40 µl 25 × HiTransG lentivirus infection reagent (GeneChem, Inc.) + 950 µl complete medium. After 16 h of infection, the medium was replaced with complete medium, and the cells were cultured for 72 h. Then, these cells were selected with 2 µg/ml of puromycin (cat. no. ab141453; Abcam) for three days and harvested for further studies.

Plasmids

A Flag-tagged PTEN wild-type (WT) plasmid [pLV3-CMV-PTEN (human)-3X FLAG-Puro; cat. no. P64887] was purchased from Wuhan MiaoLing Biotech Science Co., Ltd. PTEN S380A mutants were generated by Hieff Mut™ Site-Directed Mutagenesis Kit (cat. no. 11003ES10; Shanghai Yeasen Biotechnology Co., Ltd.). The primers used were as follows: forward, 5′-TATAGATATGCTGACACCACTGACTCTGATCCAGAGA-3′ and reverse, 5′-TGGTGTCAGCATATCTATAATGATCAGGTTCATTGTCA-3′. Transfections were conducted using Lipofectamine™ 3000 (cat. no. L3000015; Invitrogen' Thermo Fisher scientific, Inc.).

Proliferation, colony formation and 5-ethynyl-2′-deoxyuridine (EdU) assays

For the proliferation assay, RBE and HCCC-9810 cells transfected with the PTP4A1-knockdown (KD) or OE lentiviral vector (3,000 per well) were seeded into 96-well plates and treated with Cell Counting Kit-8 (CCK-8) reagent (10 µl/well; Beyotime Institute of Biotechnology) at 37°C for 2 h. Subsequently, cell viability at 24, 48 or 72 h was evaluated using a microplate reader (Thermo Fisher Scientific, Inc.) at 450 nm. Experiments were performed in triplicate.

For the colony formation assay, RBE and HCCC-9810 cells transfected with the PTP4A1-KD or OE lentiviral vector (500 per well) were seed into 6-well plates and cultured in DMEM plus 10% FBS in a 5% CO2 incubator at 37°C for 14 days and then fixed with 4% PFA for 1 h at room temperature. The cells were then stained with crystal violet (0.1%) at room temperature for 30 min. Images of the colonies were subsequently captured with an inverted light microscope with camera functionality at ×10 magnification (3 images per sample). Experiments were performed in triplicate.

For the EdU assay, 3×105 RBE and HCCC-9810 cells transfected with the PTP4A1-KD were inoculated into 24-well plates and treated with 2 µM PTEN inhibitor SF1670 (cat. no. S7310; Selleck Chemicals) or 10 µM GSK3 inhibitor SB216763 (cat. no. S1075; Selleck Chemicals) for 24 h. Then, the cells were incubated with 20 µM EdU (Guangzhou RiboBio, Co., Ltd.) at 37°C for 2 h. Subsequently, the cells were fixed and permeated with 4% PFA for 30 min and 0.5% Triton X-100 for 10 min at room temperature. After being washed with PBS, the cells were reacted with 300 µl of Apollo reaction cocktail (Guangzhou RiboBio, Co., Ltd.) for 30 min, and the cell nuclei were stained with 300 ml of 1X Hoechst 33342 (Guangzhou RiboBio, Co., Ltd.) for 30 min. Finally, the cells were analyzed by fluorescence microscopy (magnification, ×200; Nikon Corporation). Experiments were performed in triplicate.

Tumorigenesis in nude mice

A total of 10 male BALB/c nude mice (5 weeks-old; weighing 20 g) purchased from Hunan SJA Laboratory Animal Co., Ltd., were used to establish a xenograft model and were maintained in an environment at 24±2°C and 50±5% humidity with a 12/12-h light/dark cycle. The animals were fed an autoclaved laboratory rodent diet. RBE/HCCC-9810 cell lines stably transfected with PTP4A1-KD and NC lentiviral vectors (5×106 cells/mouse) in 0.1 ml PBS were subcutaneously injected into the axillae of the anterior left limbs of the mice. Tumor volume was measured every four days for 4 weeks. The tumor volume was calculated as follows: Volume=0.5 × long diameter × short diameter2. A tumor diameter exceeding 17 mm, weight loss >20% of body weight, the animal exhibiting cachexia or wasting syndrome or the size of the solid tumor being >10% of body weight were considered as humane endpoint. The animals were sacrificed if any humane endpoints were reached. In the present study, the maximum tumor volume and diameter measured in vivo were 408.35 mm3 and 12 mm respectively. Notably, none of the mice succumbed to humane endpoints during the experimental process. The mice were euthanized by cervical dislocation, in accordance with the recommendations of the American Veterinary Medical Association Guidelines for the Euthanasia of Animals. Tumors were harvested for imaging and monitored using Vernier calipers. All animal experiments described in the present study were reviewed and approved by Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University) (approval no. 2024-156). All animal protocols complied with the ARRIVE guidelines.

Wound healing assays

A total of 3×105 RBE and HCCC-9810 cells transfected with PTP4A1-KD or OE lentiviral vectors were cultured in 6-well plates for 24 h, after which an artificial scratch wound was generated with a sterile pipette tip (200 µl). The floating cells were then gently removed. The cells were subsequently cultured with serum-free medium at 37°C for 24 h. The width of each scratch was determined using an inverted microscope with camera functionality at 0 and 24 h after scratching, and the width of each wound was detected using ImageJ software (version 1.8.0; National Institutes of Health). Experiments were performed in triplicate.

Migration and invasion assays

Cell invasion and migration abilities were analyzed in Transwell assays using Transwell chambers (pore size: 8 mm, 24-well; Corning, Inc.) with or without Matrigel (BD Biosciences), respectively. Matrigel basement membrane matrix was diluted with serum-free DMEM (1:1). Matrigel matrix (100 µl/well) was added into the upper chamber and then solidified in an incubator at 37°C for 2 h. A total of 5×104 or 1×105 RBE or HCCC-9810 cells were suspended in 200 µl of serum-free medium and seeded into the upper chamber for migration and invasion, respectively. A total of 600 µl of medium plus 10% FBS was added to the lower chamber. Then the cells were treated with PTEN inhibitor SF1670 or GSK3 inhibitor SB216763. After incubation at 37°C with 5% CO2 for 24 h, the cells were fixed with 4% PFA at room temperature for 30 min and stained with 0.5% crystal violet at room temperature for 15 min. Images of the migratory and invasive cells were captured using an inverted light microscope with camera functionality. Experiments were performed in triplicate.

WB

The cells and tissues were lysed on ice in radioimmunoprecipitation assay (RIPA) lysis buffer (Shanghai Yeasen Biotechnology Co., Ltd.) supplemented with protease inhibitor cocktail tablets. Cell lysates were clarified by centrifugation (20 min at 15,000 × g at 4°C) and protein concentration determined using the BCA protein assay (Biosharp Life Sciences). Equal amounts of protein (40 µg) were subjected to 8–12% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, blocked in 10% milk at room temperature for 1 h and probed with anti-PTP4A1 (1:1,000; cat. no. 67584-1-Ig; Proteintech Group, Inc.), anti-AKT (1:1,000; cat. no. 9272S; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-AKT (1:1,000; cat. no. 4060S; Cell Signaling Technology, Inc.), anti-PI3K (1:1,000; cat. no. R22768; Zen-bio, Inc.), anti-p-PI3K (1:1,000; cat. no. 341468; Zen-bio, Inc.), anti-PTEN (1:1,000; cat. no. R381415; Zen-bio, Inc.) and anti-p-PTEN (1:1,000; cat. no. 9551; Cell Signaling Technology, Inc.) antibodies at 4°C overnight. The membranes were subsequently incubated with anti-mouse HRP-conjugated antibodies (1:2,000; cat. no. SA00001-1; Proteintech Group, Inc.) or anti-rabbit HRP-conjugated antibodies (1:2,000; cat. no. SA00001-2; Proteintech Group, Inc.) for 1 h at room temperature. An enhanced chemiluminescence (ECL; cat. no. AC13895; Acmec Biochemical) detection system was used to visualize the protein bands. Densitometric analysis of western blots was performed using Image Lab Software (version 6.1; Bio-Rad Laboratories, Inc.). Experiments were performed in triplicate.

RNA extraction and RT-qPCR

Total RNA was extracted from 1×106 cells or 0.5 g clinical samples using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, reverse transcription was performed using a PrimeScript RT Reagent Kit (cat. no. BL1018A; Biosharp Life Sciences) according to the manufacturer's protocols. The synthesized cDNA was used as a template for PCR, which was performed using an ABI 7000 quantitative PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) with a SYBR Green PCR kit (Vazyme Biotech Co., Ltd.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and annealing at 60°C for 30 sec, followed by 1 cycle of 95°C for 15 sec, 60°C for 30 sec and 95°C for 15 sec. Each reaction was performed in triplicate, and the expression values were normalized to those of the internal control GAPDH. mRNA expression was analyzed based on the 2−ΔΔCq relative quantification method (21). The primers used for amplification were as follows: PTP4A1 forward, 5′-ATTGAAGGTGGAATGAAATACGAAG-3′ and reverse, 5′-TACTTCTCCAAATACAGAAGTTGCT-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG'. Experiments were performed in triplicate.

IHC

ICC tumor tissues, matched adjacent non-tumor tissues tumor xenografts derived from HCCC-9810 and RBE cell lines were fixed in 4% PFA at room temperature for 24 h, paraffin embedded and sectioned to a thickness of ~5 µm. The embedded tissues were dewaxed with xylene (10 min × 3 times), followed by rehydration with gradient ethanol (anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 5 min each), and antigen retrieval using microwave for 15 min. The primary antibody anti-PTP4A1 (1:200), anti-Ki67 (1:200; cat. no. R381101; Zen-bio, Inc.) and its corresponding secondary antibody (1:1,000) were subsequently applied, and the samples were incubated with the tissue slides in a wet box at 37°C for 2 h. Nuclei were counterstained with hematoxylin. The cells were imaged using a light microscope with camera functionality. ImageJ software (version 1.8.0; National Institutes of Health) was then used to calculate the average value and immunoreactivity score (IRS). The percentages of positively stained cells were scored from 1–4 as follows: 0, 0–5; 1, 6–25; 2, 26–50; 3, 51–75; and 4, 76–100%. The staining intensity score was scored from 0–3 as follows: 0, no staining signals; 1, weak staining; 2, moderate staining; and 3, strong staining. The IRS was the comprehensive score obtained by multiplying the staining cell score by the staining intensity score (0–12).

Immunofluorescence (IF)

RBE cells transfected with the PTP4A OE vector were seeded on coverslips (1×103 cells/cm2). After 48 h, the cells were fixed with 4% PFA at 4°C overnight. Then, the cells were permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich; Merck KGaA) for 30 min, blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) in PBS at room temperature for 2 h, and incubated with p-PTEN and PTP4A1 antibodies (1:100) at 4°C overnight. The coverslips were subsequently incubated with the Goat Anti-Rabbit IgG/Alexa Fluor 647 (1:1,000; cat. no. HY-P80952; MedChemExpress) and Goat Anti-Mouse IgG (H+L) Cy3 (1:500; cat. no. S0012; Affinity Biosciences) for 1 h at room temperature. Finally, the slides were incubated with 1 µg/ml DAPI at room temperature for 30 min, and the cells were observed under a confocal microscope. Images from the basal plane of the cells were captured and stored as digital images. Each group was treated in triplicate.

Co-immunoprecipitation (Co-IP)

RBE cells were lysed on ice in RIPA lysis buffer supplemented with protease inhibitor cocktail tablets. A total of 1 µg of PTP4A1 antibody was added to the cell lysate, which was subsequently incubated at 4°C overnight with slow shaking. Subsequently, 10 µl of protein A agarose beads was added to the cell lysates, which were subsequently incubated overnight with the PTP4A1 antibody and then for 4 h at 4°C with slow shaking. 1 µg normal rabbit IgG (cat. no. 2729S; Cell Signaling Technology, Inc.) were included as a NC. The agarose beads were subsequently centrifuged at at 600 × g for 3 min at 4°C. The supernatant was carefully aspirated. Then, 20 µl of 2X SDS gel loading buffer was added to the beads, which were boiled for 6 min for WB. Experiments were performed in triplicate.

Statistical analysis

Data analysis was performed using SPSS 20.2 (IBM Corp.). The values represent the mean ± SD from 3 independent experiments. Unpaired Student's t-tests were performed to compare the differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

Results

PTP4A1 is upregulated and associated with invasive pathological features in ICC

To investigate the function of PTP4A1 in ICC, PTP4A1 expression was first detected in ICC tissues using WB. The results revealed that PTP4A1 is overexpressed in tumor tissues compared with adjacent normal tissues (Fig. 1A). To further verify PTP4A1 expression in ICC, the expression levels of PTP4A1 in 20 pairs of ICC tissues and matched adjacent normal tissues were analyzed using IHC. The results revealed that PTP4A1 was highly expressed in ICC tissues (Fig. 1B and C). RT-qPCR was subsequently performed to evaluate the mRNA levels of PTP4A1 in 40 pairs of ICC tumor tissues and matched adjacent normal tissues. PTP4A1 mRNA was frequently upregulated in ICC (Fig. 1D). Furthermore, the relationships between PTP4A1 expression and the clinicopathological characteristics of patients with ICC were explored. Compared with that in patients with ICC with negative lymph node metastasis, PTP4A1 mRNA expression was higher in patients with ICC with positive lymph node metastasis (Fig. 1E). In addition, the PTP4A1 mRNA was overexpressed in patients with ICC with vascular invasion (Fig. 1F). Compared with patients with I/II TNM stage ICC, PTP4A1 was highly expressed in patients with III/IV TNM stage ICC (Fig. 1G). Moreover, the expression of PTP4A1 was related to the differentiation of ICC. PTP4A1 mRNA expression in poorly differentiated ICC tissues was higher than the levels in well-differentiated and moderately differentiated ICC tissues (Fig. 1H). Collectively, these results indicated that PTP4A1 is overexpressed and associated with aggressive pathological characteristics in ICC.

Figure 1. PTP4A1 is highly expressed and associated with aggressive pathological characteristics in ICC. (A) PTP4A1 expression levels in 10 pairs of ICC tissues (T) and matched adjacent normal tissues (N). (B) Representative images of IHC staining for PTP4A1 in ICC tumor tissues and matched adjacent normal tissues. (C) PTP4A1 IHC scores in ICC tumor tissues and matched adjacent normal tissues. (D) PTP4A1 mRNA levels were greater in ICC tissues than in adjacent normal tissues. (E-H) Higher mRNA expression of PTP4A1 was positively associated with (E) lymph node metastasis, (F) vascular invasion, (G) III/IV TNM stage and (H) poor differentiation in ICC. *P<0.05 and **P<0.01. PTP4A1, protein tyrosine phosphatase 4A1; ICC, intrahepatic cholangiocarcinoma; IHC, immunohistochemical.

PTP4A1 promotes ICC cell proliferation, migration and invasion in vitro and in vivo

To investigate the potential biological function of PTP4A1 in ICC, RBE and HCCC-9810 cells were stably transfected with PTP4A1 KD and OE lentiviruses. The expression levels of PTP4A1 in the ICC cell lines RBE and HCCC-9810 were analyzed using WB and RT-qPCR. As demonstrated in Fig. 2A and B, the expression of PTP4A1 was significantly decreased in RBE and HCCC-9810 cells transfected with PTP4A1 KD lentivirus and significantly increased in REB and HCCC-9810 cells transfected with PTP4A1-OE lentivirus compared with that in ICC cells transfected with NC or EV lentivirus. CCK-8, EdU and colony formation assays were subsequently performed to evaluate the role of PTP4A1 in the proliferation of ICC cells. The results of the CCK-8 assays suggested that PTP4A1 promoted the proliferation of RBE and HCCC-9810 cells (Fig. 2C and D). The results of the EdU assays demonstrated that the viabilities of the RBE and HCCC-9810 cells transfected with the PTP4A1-KD lentivirus were lower than those of the corresponding NC cells (Fig. 2E and F). Consistent with these findings, the viabilities of RBE and HCCC-9810 cells transfected with the PTP4A1-OE lentivirus were greater than those of the corresponding EV-transfected cells (Fig. 2E and F). Additionally, the results of the colony formation assay revealed that PTP4A1 increased the survival ability of RBE and HCCC-9810 cells (Fig. 2G and H). Furthermore, a subcutaneous tumor formation experiment was conducted in nude mice in which RBE and HCCC-9810 cells were transfected with PTP4A1-KD lentivirus or NC lentivirus. All the mice successfully formed tumors. ICC tumor tissue images revealed that the tumors that originated from RBE and HCCC-9810 cells transfected with the PTP4A1-KD lentivirus were smaller than those in the NC groups (Fig. 2I). The tumor growth curves revealed that the tumors in RBE and HCCC-9810 cells transfected with PTP4A1-KD lentivirus grew slower than those in the NC groups did at the same time points (Fig. 2J). The volume of tumor tissues derived from RBE and HCCC-9810 cells transfected with the PTP4A1-KD lentivirus was significantly smaller than that derived from the NC cells (Fig. 2K and L). The IHC results revealed that the expression of the proliferation marker Ki67 was decreased in tumor tissues derived from RBE and HCCC-9810 cells transfected with the PTP4A1-KD lentivirus (Fig. 2N). Collectively, these results indicated that PTP4A1 promotes ICC proliferation in vitro and in vivo.

Figure 2. PTP4A1 promotes intrahepatic cholangiocarcinoma cell proliferation in vitro and in vivo. (A) Western blotting and (B) reverse transcription-quantitative PCR were performed to detect PTP4A1 mRNA and protein expression levels in RBE and HCCC-9810 cells stably transfected with PTP4A1-OE or PTP4A1-KD lentivirus. (C and D) Cell Counting Kit-8, (E and F) EdU and (G and H) cell colony formation assays were used to evaluate the proliferation and viability of RBE and HCCC-9810 cells stably transfected with PTP4A1-OE or PTP4A1-KD lentivirus. (I) RBE and HCCC-9810 cells stably transfected with PTP4A1-KD lentivirus and NC cells were injected into nude mice, and subcutaneous xenograft tumors were successfully established (n=5 for each group). (J) Compared with those in the NC group, tumor weight were significantly decreased in the PTP4A1-KD group. (K and L) The tumor growth curves of RBE (K) and HCCC-9810 (L) cells in nude mice. (M) Representative immunohistochemical staining for Ki67 in xenograft tumor tissues. *P<0.05 and **P<0.01. PTP4A1, protein tyrosine phosphatase 4A1; OE, overexpression; KD, knockdown; NC, negative control; EV, empty vector.

To further explore the function of PTP4A1 in ICC metastasis, wound healing assays were performed, and the results revealed that PTP4A1-OE increased the migration of RBE and HCCC-9810 cells. However, the migration abilities of PTP4A1-downregulated RBE and HCCC-9810 cells were reduced (Fig. 3A and B). Similarly, the results of migration assays demonstrated that PTP4A1 promoted the migration of RBE and HCCC-9810 cells (Fig. 3C and D). Invasion assays suggested that PTP4A1 increased invasion in RBE and HCCC-9810 cells (Fig. 3E and F). In addition, PTP4A1-OE promoted epithelial-mesenchymal transition (EMT), and PTP4A1-KD inhibited EMT in RBE and HCCC-9810 cells (Fig. 3G-N). Furthermore, the results showed that PTP4A1-OE significantly increases the expression levels of MMP2 and MMP9, which are key enzymes involved in tumor invasion and metastasis. Conversely, PTP4A1-KD leads to a decrease in MMP2 and MMP9 expression (Fig. 3G-N). Expression of the epithelial cell marker E-cadherin was decreased and that of the mesenchymal cell marker N-cadherin was increased in tumorigenic tissues derived from RBE and HCCC-9810 cells transfected with PTP4A1-KD lentivirus (Fig. 3M). These results demonstrated that PTP4A1 promoted migration, invasion and EMT in ICC.

Figure 3. PTP4A1 promotes migration, invasion and EMT in intrahepatic cholangiocarcinoma. (A and B) Wound healing and (C and D) migration assays were used to detect the effect of PTP4A1 on the migration of RBE and HCCC-9810 cells. Invasion assays (E and F) were used to evaluate the invasion abilities of RBE and HCCC-9810 cells stably transfected with PTP4A1-overexpressing and PTP4A1-knockdown lentiviruses. (G) EMT marker and MMP2/MMP9 expression in RBE and HCCC-9810 cells stably transfected with PTP4A1-OE and PTP4A1-KD lentiviruses. (H-N) Quantitative analysis of (H) FN1, (I) E-cadherin, (J) N-cadherin, (K) Vimetin, (L) MMP9 and (N) MMP2 expression levels. (M) E-cadherin and N-cadherin expression levels in tumorigenic tissues were measured by immunohistochemistry. **P<0.01. PTP4A1, protein tyrosine phosphatase 4A1; EMT, epithelial-mesenchymal transition; OE, overexpression; KD, knockdown; NC, negative control; EV, empty vector.

PTP4A1 interacts with PTEN and is involved in the activation of the PI3K/AKT/GSK3α pathway

To further clarify the mechanism by which PTP4A1 promotes the progression of ICC, Co-IP-MS was performed to screen for proteins that interact with PTP4A1. A Venn diagram revealed that 87 proteins may interact with PTP4A1 (Fig. 4A). Among them, focus was addressed on PTEN. To further verify the interaction between PTP4A1 and PTEN, IF staining was performed. The results revealed that PTP4A1 colocalized with PTEN in RBE cells (Fig. 4B). Co-IP analysis verified that PTP4A1 interacted with PTEN in RBE cells transfected with PTP4A1-OE lentivirus (Fig. 4C and D). PTP4A1 belongs to the PTP family and may play a role in the dephosphorylation of its interacting proteins. Next, the effect of PTP4A1 on PTEN phosphorylation was evaluated. The results revealed that PTP4A1-OE decreased PTEN phosphorylation and that KD of PTP4A1 increased the phosphorylation level of PTEN in RBE and HCCC-9810 cells, indicating that PTP4A1 inhibited the activation of PTEN (Fig. 4E). PTEN is a negative regulator of the PI3K/AKT/GSk3α signaling pathway. The activation of PI3K/AKT/GSk3α signaling in PTP4A1-OE and PTP4A1-KD RBE and HCCC-9810 cells was subsequently detected (Fig. 4E). These results indicated that PTP4A1 contributes to the activation of the PI3K/AKT/GSk3α signaling pathway by interacting with and regulating PTEN.

Figure 4. PTP4A1 interacts with PTEN and regulates the activation of the PI3K/AKT/GSKα pathway. (A) Venn diagram showing the potential PTP4A1-interacting proteins screened by co-IP-MS based on the screening criteria of ‘1-PTP4A1-flag and 2-PTP4A1-flag not 1-PTP4A1-IgG not 2-PTP4A1-IgG’. (B) PTP4A1 colocalized with PTEN in RBE cells. (C and D) Co-IPs were performed to confirm the association between PTP4A1 and PTEN. (E) PTP4A1 regulated the activation of the PTEN/PI3K/AKT/GSKα signaling pathway in RBE and HCCC-9810 cells. PTP4A1, protein tyrosine phosphatase 4A1; PTEN, phosphatase and tensin homolog; co-IP, co-immunoprecipitation; OE, overexpression; KD, knockdown; NC, negative control; EV, empty vector; p-, phosphorylated.

To further validate the inhibitory role of PTP4A1 in PTEN phosphorylation, a phospho-mimetic PTEN mutant plasmid (PTEN S380A) was first constructed and transfected into RBE and HCCC-9810 cells. The results of WB showed the successful OE of PTEN S380A (Fig. 5A). Subsequently, PTEN S380A plasmid was overexpressed in RBE and HCCC-9810 cells with stable PTP4A1-OE. WB revealed that the phospho-mimetic PTEN S380 mutant markedly diminished the phosphorylation levels of PI3K and AKT (Fig. 5B). Additionally, mimetic phosphorylation of PTEN S380 attenuated the PTP4A1-driven promotion of cell proliferation (Fig. 5C and D), invasion (Fig. 5E and F) and metastasis (Fig. 5G and H) in RBE and HCCC-9810 cells. These findings suggest that PTP4A1 may exert its oncogenic effects by modulating the PTEN/PI3K/AKT signaling pathway.

Figure 5. Inhibitory role of PTP4A1 on PTEN phosphorylation. (A) PTEN S380A was overexpressed in the RBE and HCCC-9810 cells. (B) Western blotting was used to detect the expression and phosphorylation of PI3K and AKT and in PTP4A-OE RBE and HCCC-9810 cells transfected with the phospho-mimetic PTEN mutant plasmid (PTEN S380A). (C-H) Phospho-mimetic PTEN S380A reversed the promoting effects of PTP4A1 on the (C and D) proliferation, (E and F) invasion and (G and H) metastasis of RBE and HCCC-9810 cells. **P<0.01. PTP4A1, protein tyrosine phosphatase 4A1; PTEN, phosphatase and tensin homolog; OE, overexpression; EV, empty vector.

PTP4A1 promotes ICC progression through regulating the PTEN/PI3K/AKT/GSK3α signaling pathway

PTP4A1 promoted cell proliferation, migration and invasion; interacted with PTEN; and contributed to the activation of the PI3K/AKT/GSk3α signaling pathway. In addition, the PI3K/AKT/GSk3α pathway is involved in tumor progression. Thus, it was hypothesized that the promotion of cell proliferation, invasion and metastasis by PTP4A1 is dependent on the regulation of the PTEN/PI3K/AKT/GSk3α pathway. To test this hypothesis, RBE and HCCC-9810 cells were stably transfected with PTP4A1-KD lentivirus with the PTEN inhibitor SF1670 or the GSK3α inhibitor SB216763. The WB results revealed that the KD of PTP4A1 increased PTEN phosphorylation and decreased the activation of the PI3K/AKT/GSk3α pathway in RBE and HCCC-9810 cells (Fig. 6A). Additionally, the PTEN inhibitor SF1670 significantly inhibited PTEN phosphorylation and activated the PI3K/AKT/GSk3α pathway in RBE and HCCC-9810 cells with PTP4A1 KD (Fig. 6A). As a downstream molecule of PI3K/AKT, the GSK3α inhibitor SB216763 did not affect the phosphorylation of PTEN, PI3K or AKT (Fig. 6A). EdU assays revealed that the PTEN inhibitor SF1670 and the GSK3α inhibitor SB216763 significantly attenuated the inhibitory effect of PTP4A1 KD on the proliferation of RBE and HCCC-9810 cells (Fig. 6B and C). Furthermore, the results of migration (Fig. 6D and E) and invasion assays (Fig. 6F and G) revealed that the PTEN inhibitor SF1670 and the GSK3α inhibitor SB216763 reversed the inhibitory effects of PTP4A1 KD on the migration and invasion of RBE and HCCC-9810 cells. To validate the off-target effects of the PTEN inhibitor SF1670 and GSK3α inhibitor SB216763 on other pathways (ERK and TGF-β), the ERK and TGF-β signaling pathways were evaluated in RBE and HCCC-9810 cells co-treated with SF1670 or SB216763 and TGF-β. The results demonstrated that SF1670 and SB216763 did not significantly affect ERK phosphorylation levels or the activation status of the TGF-β signaling pathway in these cells, suggesting that these inhibitors did not exhibit off-target effects (Fig. 6H). Collectively, these results indicate that PTP4A1 promotes the progression of ICC by regulating the PTEN/PI3K/AKT/GSk3α pathway.

Figure 6. PTP4A1 promotes intrahepatic cholangiocarcinoma cell proliferation, migration and invasion by regulating the PTEN/PI3K/AKT/GSk3α pathway. (A) WB was performed to examine the expression and phosphorylation of PTEN, PI3K, AKT and GSk3α in PTP4A-KD RBE and HCCC-9810 cells treated with the PTEN inhibitor SF1670 or the GSK3α inhibitor SB216763. (B and C) A 5-ethynyl-2′-deoxyuridine assay was used to evaluate the effects of the PTEN inhibitor SF1670 or the GSK3α inhibitor SB216763 on the proliferation of PTP4A-KD RBE and HCCC-9810 cells. (D-G) SF1670 and SB216763 attenuated the inhibitory effects of PTP4A1 knockdown on (D and E) migration and (F and G) invasion in RBE and HCCC-9810 cells. (H) WB was used to evaluate the ERK and TGF-β signaling pathways in RBE and HCCC-9810 cells co-treated with SF1670 or SB216763 and TGF-β. **P<0.01. PTP4A1, protein tyrosine phosphatase 4A1; PTEN, phosphatase and tensin homolog; WB, western blotting; KD, knockdown; NC, negative control; p-, phosphorylated.

Discussion

ICC is a highly aggressive tumor with an advanced clinical stage, limited therapeutic options, and a high postoperative recurrence rate (22,23). Moreover, the prognosis of ICC is extremely poor, with a 5-year survival rate of only 25–40% (5); moreover, the median overall survival (OS) time of patients with advanced ICC is as low as ~22 months (24,25). The molecular pathogenesis of ICC is complex and not fully understood. A deeper understanding of the molecular mechanisms of ICC may contribute to the development of novel therapeutic approaches to improve ICC prognosis. In the present study, it was revealed that PTP4A1 was overexpressed and associated with aggressive clinicopathological characteristics in ICC. Furthermore, it was demonstrated that PTP4A1 promoted cell proliferation, migration and invasion by interacting with PTEN and activating the PI3K/AKT/GSK3α pathway.

Accumulating data suggest that PTP4A1 functions as an oncogene and is involved in tumor progression (26). An increasing number of studies, including the authors' previous study (20), have revealed that the OE of PTP4A1 is associated with poor OS in various types of tumors, such as NSCLC (9), oral squamous cell carcinoma (OSCC) (18), cervical cancer (27) and HCC (16,19,28). Similarly, it was revealed that PTP4A1 was highly expressed in ICC tissues. In addition, the present study demonstrated that PTP4A1 is associated with lymph node metastasis, vascular invasion, advanced TNM stage and poor differentiation of ICC. Consistent with the present findings, overexpressed PTP4A1 was significantly correlated with advanced TNM stage in patients with HCC (16,19) and with lymphatic metastasis in patients with NSCLC (9). Based on the aforementioned results, it was hypothesized that PTP4A1 plays an essential role in tumor progression.

Furthermore, the findings of the present study showed that PTP4A1 KD decreased ICC cell proliferation, migration and invasion in vivo and in vitro. Similarly, PTP4A1 OE increased these processes, which is consistent with the findings of previous studies in colorectal cancer (17,29), OSCC (18), esophageal squamous cell carcinoma (ESCC) (30) and HCC (16,19). Mechanistically, a previous study indicated that PTP4A1 regulated proliferation and apoptosis by downregulating the protein level of P53 (17) and promoted EMT via the ERK/GSK3β/β-catenin pathway in colon cancer (29). In cervical cancer, PTP4A1 aggravated malignant progression by activating the ERK pathway (13,31). Additionally, PTP4A1 contributes to metastasis through altering mitochondrial metabolic reprogramming mediated by PMK2 and ACO2 in OSCC (18). These results indicate that PTP4A1 exerts oncogenic effects through diverse mechanisms across distinct tumor types. However, it has been reported that PTP4A1 promotes migration and invasion by activating the PI3K/AKT pathway in HCC (16,19) and ESCC (30). ICC, a liver malignancy with a relatively low incidence rate, shares similarities with HCC in terms of oncogenesis.

PTP4A1 has been reported to suppress E-cadherin and increase migration and invasion via the PI3K/AKT and ERK pathways in HCC (16,19), promote TGF-β signaling in human fibroblasts through interacting with SRC and activating the ERK pathway (15).

The present study demonstrated that PTP4A1 promoted cell viability, migration and invasion by activating the PI3K/AKT pathway in ICC. Nevertheless, the molecular mechanism by which PTP4A1 activates the PI3K/AKT pathway remains unclear. PTEN is a classical tumor suppressor that antagonizes PI3K/AKT signaling (32,33). In the present study, it was found that PTP4A1 interacted with PTEN. PTP4A1 is a protein tyrosine phosphatase, and its OE decreased the phosphorylation of PTEN and increased the phosphorylation of PI3K, AKT and its downstream protein GSK3α. By contrast, PTP4A1 KD increased PTEN phosphorylation and inactivated PI3K/AKT/GSK3α, indicating that PTP4A1 activated the PI3K/AKT pathway by suppressing the activation of PTEN. In addition, the PTEN inhibitor SF1670 and the GSK3α inhibitor SB216763 significantly attenuated the inhibitory effects of PTP4A1 KD on the proliferation, migration and invasion of ICC cells.

In conclusion, the present study demonstrated that PTP4A1 was overexpressed and associated with aggressive pathological characteristics in ICC. As an oncogene, PTP4A1 promotes cell proliferation, migration and invasion in a manner dependent on the regulation of the PTEN/PI3K/AKT/GSk3α pathway, indicating that PTP4A1 may serve as a valuable therapeutic target for ICC.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Fund of Science and Technology Department of Hunan (grant no. 2018SK50726).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

OL was responsible for project development, data collection and manuscript writing. YP collected and analyzed data. JC collected data. YL contributed to the study conception and design, revised and edited the manuscript. All authors read and approved the final version of the manuscript. OL and YP confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The experimental protocols were approved (approval no. 2023-151) by the Ethics Committee of Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University; Changsha, China). Written informed consent was obtained from all participants. All animal experiments described in the present study were reviewed and approved by Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University) (approval no. 2024-156). All animal protocols complied with the ARRIVE guidelines. Euthanasia was performed by means of cervical dislocation, in accordance with the recommendations of the American Veterinary Medical Association Guidelines for the Euthanasia of Animals.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References