TIPE3 promotes breast cancer progression and metastasis via the AKT-GSK3β-β-catenin/Snail pathway

Introduction

Breast cancer is the most commonly diagnosed malignant tumor and is the leading cause of cancer-related mortality among women worldwide (1). It is also one of the most prevalent malignancies among women in China, with an increasing incidence rate and a trend toward younger onsets (2). Currently, the main treatment modalities for breast cancer include surgery, radiotherapy, chemotherapy, endocrine therapy, and molecular targeted therapy (3). However, both primary and acquired therapeutic resistance, along with the limited durability of treatment efficacy, represent major challenges in breast cancer management. Resistance to conventional treatment, recurrence and metastasis of breast cancer are the primary factors leading to the failure of breast cancer treatment. Therefore, investigating the molecular mechanisms underlying breast cancer metastasis and identifying novel, effective therapeutic targets for intervention are of great significance for improving the efficacy of breast cancer and its prognosis.

The tumor necrosis factor-α-induced protein 8 (TNFAIP8, TIPE) family is a recently identified group of genes associated with inflammation and tumorigenesis, which has garnered increasing attention for its role in cancer progression (4-6). To date, this family consists of four identified members: TNFAIP8 (TIPE), TNFAIP8L1 (TIPE1), TNFAIP8L2 (TIPE2), and TNFAIP8L3 (TIPE3) (4-6). Existing studies indicate that TIPE functions as an oncogene, exhibiting high expression in various human cancer cells and promoting tumor growth and metastasis (4,5). The role of TIPE1 in tumor progression remains largely unexplored, however, studies in hepatocellular carcinoma (HCC) models suggest that TIPE1 negatively regulates Rac1, thereby inhibiting HCC cell growth and inducing apoptosis, implying that TIPE1 may function as a tumor suppressor gene (7). TIPE2 is a novel death effector domain (DED)-containing protein that is selectively highly expressed in lymphoid and myeloid cells and serves as a negative regulator of immune responses. It plays a crucial role in maintaining homeostasis in both innate and adaptive immunity (4-6,8,9) and exhibits dual tumor-suppressive and tumor-promoting properties in cancer (4,5). TIPE2 inhibits the proliferation, migration, and invasion of gastric, breast, and prostate cancer cells by suppressing the protein kinase B (AKT), extracellular regulated protein kinases (ERK1/2), and p38 signaling pathways (10-13). TIPE3 acts as a novel lipid transporter for the second messengers phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2, PIP2] and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3], participating in phosphoinositide transport and metabolism to facilitate the activation of phosphatidylinositol 3-kinase (PI3K)-AKT signaling (14,15). TIPE3 exhibits high expression levels in esophageal cancer, cervical cancer, colon cancer, lung cancer and various other malignancies (14), indicating that TIPE3 could potentially function as an oncogene that promotes tumorigenesis and progression. Cell membrane-localized TIPE3 also facilitates lung cancer growth and migration by activating AKT and ERK signaling (16). In breast cancer, Lian et al. reported that TIPE3 upregulates matrix metalloproteinase 2 (MMP2) and urokinase type plasminogen activator (uPA) levels while activating AKT and nuclear transcription factor-κB (NF-κB) signaling pathways, suggesting that TIPE3 may promote breast cancer growth and metastasis through these mechanisms and function as a potential biomarker for breast cancer metastasis (17). However, the precise role and molecular mechanisms of TIPE3 in breast cancer remain largely unclear.

The PI3K-AKT signaling pathway is critical for regulating cell growth, and it is widely acknowledged for its significant involvement in tumorigenesis and development. Abnormal activation of AKT signaling in tumor cells can drive malignant cell cycle progression, promote survival, resist apoptosis and participate in uncontrolled tumor growth, invasion and metastasis (18-20). AKT can inactivate glycogen synthase kinase 3β (GSK3β) activity by direct phosphorylation of GSK3β, inhibit GSK3β phosphorylation of β-catenin, block proteasomal degradation of β-catenin, thereby upregulating β-catenin nuclear transcriptional signaling (21-23). β-catenin, as one of the key regulatory molecules in the classical Wnt signaling pathway, significantly influences embryonic development, tissue homeostasis, stem cell formation, cell proliferation, differentiation, and motility, and its overactivation is closely related to cancer and other diseases (24-29). Furthermore, epithelial-mesenchymal transition (EMT), as a developmental regulatory process, plays a crucial role in tumor cell migration, invasion and metastasis (30-41). EMT is a key biological process that enables malignant epithelial-derived tumor cells to acquire metastatic potential (30-41). It is characterized by the downregulation of the epithelial marker E-cadherin and the upregulation of mesenchymal marker N-cadherin and the cytoskeletal protein Vimentin (30-41).

Therefore, this study aims to construct breast cancer transgenic cells with TIPE3 overexpression and knockdown, and use both cellular and animal models to investigate the role of TIPE3 in breast cancer cell growth and metastasis. This study seeks to elucidate the molecular mechanism by which TIPE3 regulates the AKT-GSK3β-β-catenin/Snail axis to promote tumor growth and metastasis in breast cancer. It is anticipated that TIPE3 may serve as a potential therapeutic target for breast cancer, providing new insights into its prevention and treatment. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-717/rc).

Methods

Cell culture

The normal human breast epithelial cell lines MCF10A and human breast cancer cell lines including MCF7, SKBR3, MDA-MB-231 and MDA-MB-468 purchased from the Cell Resource Center of Chinese Academy of Science Committee (Shanghai, China), were cultured in DMEM growth medium (Hyclone, UT, USA) with 10% fetal bovine serum (FBS) (Gibco, MD, USA) and 100 U/mL penicillin-streptomycin solution (Beyotime, Beijing, China) at 37 ℃ in humidified 5% CO₂.

Collection of breast cancer specimens of breast cancer patients

Neutral formalin (10%)-fixed, paraffin-embedded and frozen 100 pairs of breast cancer tissue and adjacent nontumor tissue sections from 100 patients with operable breast cancer (all the enrolled patients did not receive any preoperative treatments) were provided by Breast Surgery of The First Affiliated Hospital of Soochow University. All participating patients signed an informed consent form, and the study was approved by the Medical Ethics Committee of The First Affiliated Hospital of Soochow University (IRB No. 2021-036). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Immunohistochemistry (IHC) staining and scoring

Each section was analyzed by IHC according to the manufacturer’s instructions, the localization of cytoplasm, membrane and nucleus was determined, and then IHC scoring was performed according to the proportion of positively stained cells and intensity of staining as previously described (42). The expression level of TIPE3 in breast cancer tissues was analyzed according to the immunohistochemical scores of TIPE3 in breast cancer tissues and adjacent tissues (−, +, ++, +++). Breast cancer patients were divided into two groups: high TIPE3 expression group (++, +++) and low TIPE3 expression group (−, +). TIPE3 expression level in breast cancer tissues was analyzed.

Establishment of TIPE3 overexpression and knockdown breast cancer cell lines

Lentivirus (LV) [lentiviral plasmid: pLenti6.3/short hairpin RNA (shRNA)/green fluorescent protein (GFP), packaging plasmids: pLP1, pLP2, and vesicular stomatitis virus glycoprotein (VSVG)] carrying human TIPE3 shRNA-TIPE3 1# (shRNA-TIPE3-541-F, 5'-CAC CGC TCT ACA AAG TCA CCA AAG ACG AAT CTT TGG TGA CTT TGT AGA GC-3'; shRNA-TIPE3-541-R, 5'-AAA AGC TCT ACA AAG TCA CCA AAG ATT CGT CTT TGG TGA CTT TGT AGA GC-3'), shRNA-TIPE3 2# (shRNA-TIPE3-657-F, 5'-CAC CGC CAA GAG GAG CTG GTT ATT GCG AAC AAT AAC CAG CTC CTC TTG GC-3'; shRNA-TIPE3-657-R, 5'-AAA AGC CAA GAG GAG CTG GTT ATT GTT CGC AAT AAC CAG CTC CTC TTG GC-3'), shRNA-TIPE3 3# (shRNA-TIPE3-841-F, 5'-CAC CGC GCA TCA ACC ACG TCT TTA ACG AAT TAA AGA CGT GGT TGA TGC GC-3'; shRNA-TIPE3-841-R, 5'-AAA AGC GCA TCA ACC ACG TCT TTA ATT CGT TAA AGA CGT GGT TGA TGC GC-3') or control shRNA (shcontrol) (shcontrol-F: 5'-CAC CGG AGT GGA TCG CCG TTG ATA ACG AAT TAT CAA CGG CGA TCC ACT CC-3'; shcontrol-R: 5'-AAA AGG AGT GGA TCG CCG TTG ATA ATT CGT TAT CAA CGG CGA TCC ACT CC-3'), LV (lentiviral plasmid: pLenti6.3/IRES/GFP, packaging plasmids: pLP1, pLP2, and VSVG) carrying the TIPE3 coding sequence (CDS) and blank control LV were supplied by Novobio (Shanghai, China). All of the above LVs expressed GFP and blasticidin S deaminase. MCF7 breast cancer cell line with low relative TIPE3 expression and low invasion and migration ability was infected with LV-TIPE3 or blank control LV. MDA-MB-231 breast cancer cell line with high relative TIPE3 expression and high invasion and migration ability was infected with LV-shTIPE3 1#, LV-shTIPE3 2#, LV-shTIPE3 3#, or LV-shcontrol. All the cell lines were selected with blasticidin S. Fluorescence microscopy was used to detect the transgenic efficiency mediated by LV. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the overexpression and knockdown efficiency of TIPE3.

Western blot analysis

The protein concentration of cell lysates was determined using the BCA protein concentration determination kit (Beyotime, Beijing, China) according to the manufacturer’s instructions. Protein was separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membranes were blocked with 5% non-fat milk in tris buffered saline with Tween 20 (TBST) for 1 hour, incubated with primary specific antibodies overnight at 4 ℃, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:10,000). Protein expression levels were detected by enhanced chemiluminescence (ECL) (Millipore, MA, USA) with a Bio-Imaging System. The primary antibodies were as follows: anti-TIPE3 (1:1,000), anti-p-AKT (T308) (1:1,000), anti-p-AKT (S473) (1:1,000), anti-AKT (1:1,000), anti-p-GSK3β (1:1,000), anti-GSK3β (1:1,000), anti-β-catenin (1:1,000), anti-Snail1 (1:1,000), anti-Slug (1:1,000), anti-cyclin D1 (1:1,000), anti-E-cadherin (1:1,000), anti-N-cadherin (1:1,000), anti-vimentin (1:1,000), anti-β-actin (1:3,000), and anti-histone H3 (1:2,000). All the antibodies are supplied by Cell Signaling Technology (MA, USA).

qRT-PCR

Total RNA was extracted using the MiniBEST Universal RNA Extraction Kit (Takara, Japan) according to the manufacturer’s instructions. The first-strand cDNA synthesis was conducted through All-In-One 5X RT MasterMix Kit (ABM, BC, Canada). The quantitative real-time analysis was performed using a BlasTaq 2X qPCR MasterMix Kit (ABM, BC, Canada). The primers used were as follows: TIPE3 (TIPE3-F1, 5'-AGG ACC TGG TGC ATG AAC TG-3'; TIPE3-R1, 5'-TGA GGT TGG GCC TAC AGT CT-3'), cyclin D1 (cyclin D1-F, 5'-GAT GCC AAC CTC CTC AAC GA-3'; cyclin D1-R, 5'-GGA AGC GGT CCA GGT AGT TC-3'), E-cadherin (E-cadherin-F, 5'-TGG GCC AGG AAA TCA CAT CC-3'; E-cadherin-R, 5'-CCC CGT GTG TTA GTT CTG CT-3'), N-cadherin (N-cadherin-F, 5'-CGG CCC GCT ATT TGT CAT CA-3'; N-cadherin-R, 5'-TGC GAT TTC ACC AGA AGC CT-3'), vimentin (vimentin-F, 5'-CGG CGG GAC AGC AGG-3'; vimentin-R, 5'-GAA GCG GTC ATT CAG CTC CT-3'), β-actin (β-actin-F, 5'-CTC ACC ATG GAT GAT GAT ATC GC-3'; β-actin-R, 5'-AGG AAT CCT TCT GAC CCA TGC-3') (Sangon Biotech, Shanghai, China). The relative expression was analyzed by the 2^−ΔΔCt method described previously (43).

Cell Counting Kit-8 (CCK-8) assay

The Cell Counting Kit-8 (Beyotime, Beijing, China) was used to detect the cell proliferation, 1×10⁴ cells/200 µL per well were seeded into 96-well plates and cultured 4 days, 10 µL CCK-8 solution was added every 24 hours. After 4 hours incubation, the optical density (OD) value of each well was measured at a test wavelength of 450 nm using a microplate reader.

Colony formation assay

Cells were cultured in 6-well plates for 2 to 3 weeks. The colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Colonies were counted and the clonogenic ability of the cells was analyzed.

Cell cycle analysis

Cells were washed with phosphate-buffered saline (PBS) and fixed with 70% ethanol at 4 ℃ overnight. Before detection, cells were washed and stained with 500 µL propidium iodide (PI)/RNase Staining Buffer (BD Biosciences, CA, USA) and incubated for 5 minutes in the dark at room temperature. The cell cycle was analyzed by flow cytometry with FlowJo software.

Wound healing assay

The cells were seeded and grown in 6-well plates at 5×10⁵ cells/5 mL culture medium per well. When the cells reached confluency, scratches were made and the scraped cells were washed with PBS. The wells were loaded with the Dulbecco’s Modified Eagle Medium (DMEM) medium containing 2% FBS to maintain cell culture. The scratched areas were photographed under microscope at 0, 12, 24, 36, and 48 hours, and the migratory ability of the cells was analyzed.

Transwell invasion assay

Transwell invasion assay was conducted using 24-well transwell inserts with an 8-µm pore size (Merck Millipore, MA, USA), and the inserts were coated with Matrigel (BD Biosciences, CA, USA). Cells (1× 10⁴) in 100 µL serum-free medium were seeded in the upper transwell chamber, then the lower chambers were filled with 600 µL medium containing 10% FBS. The invaded cells after being cultured for 24 hours were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and counted. The invasive ability of the cells was analyzed.

Animal models

Tumor growth model

MDA-MB-231-shTIPE3 vs. MDA-MB-231-shcontrol breast cancer cells (2×10⁶) in 100 µL PBS were injected subcutaneously into 12 BALB/c-nude mice at 4 weeks of age (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China). All the experimental nude mice were randomly assigned into two groups for the injection of MDA-MB-231-shTIPE3 cells and MDA-MB-231-shcontrol cells, respectively. Tumors volume was calculated with the formula: volume = (width²× length)/2. At 4 weeks after inoculation, the xenograft tumors were dissected and weighed.

Tumor metastasis model

Cells (2×10⁶) in 200 µL PBS were injected into mice via the tail vein. At the 4th week after injection, all mice were sacrificed. The lung tissues of mice were fixed in 10% neutral formalin, embedded in paraffin, and sectioned, and hematoxylin and eosin (HE) staining was performed to detect lung metastasis. By monitoring tumor volume, weight and lung metastasis, the effects of TIPE3 on the growth of breast cancer cells and lung metastasis ability in nude mice were analyzed. Animal experiments were performed under a project license (IRB No. A201809059) granted by Laboratory Animal Center of Soochow University, in compliance with its institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Statistical analysis

All numerical data are expressed as mean ± standard deviation (SD) or percentage. Student’s t-test, Mann-Whitney U, Pearson’s χ² test and one-way analysis of variance (ANOVA) were used to analyze the differences between groups by SPSS 23.0. P<0.05 was considered statistically significant. All experiments were repeated at least three times.

Results

TIPE3 expression was elevated in breast cancer tissues and cells

To investigate the expression of TIPE3 in breast cancer tissues, 100 pairs of breast cancer tissues and adjacent tissues were examined by IHC assay. IHC analysis showed that TIPE3 was significantly more highly expressed in breast cancer tissues than in adjacent nontumor tissues (Figure 1A). The TIPE3 high expression group (71%; 71 cases, 33 cases scored ‘+++’ and 38 cases scored ‘++’) in breast cancer tissues was significantly larger than in adjacent nontumor tissues (27%; 27 cases, 8 cases scored ‘+++’ and 19 cases scored ‘++’) (P<0.05) (Figure 1B,1C), which demonstrated that TIPE3 was upregulated in breast cancer tissues. To further verify the expression of TIPE3 in breast cancer cells, qRT-PCR and western blot were performed to analyze the expression levels of TIPE3 in luminal breast cancer cell (MCF7), HER2⁺ breast cancer cell (SKBR3), triple negative breast cancer cell (MDA-MB-231, MDA-MB-468) and normal mammary epithelial cell MCF10A (control). As shown in Figure 1D-1F, compared with MCF10A, the breast cancer cell lines showed higher expression levels of TIPE3 (P<0.05), and the expression level of TIPE3 in luminal, triple negative and HER2⁺ breast cancer cells showed an ascending trend. The up-regulation of TIPE3 expression in breast cancer cells was consistent with the results of TIPE3 expression in breast cancer tissues.

Figure 1 TIPE3 expression was elevated in breast cancer tissues and cells. (A) Representative images of TIPE3 expression detected by IHC in breast cancer tissues. (B) TIPE3 immunohistochemical score, compared to adjacent nontumor tissue. (C) The percentage of high and low expression of TIPE3 in breast cancer tissues. N is adjacent nontumor tissue, T is tumor tissue. (D) Relative mRNA expression levels of TIPE3 in breast cancer cell lines compared with MCF10A normal mammary epithelial cell line detected by qRT-PCR. (E) Representative western blot images of TIPE3 protein expression in breast cancer cell lines. (F) Relative protein expression levels of TIPE3 in breast cancer cell lines compared with MCF10A normal mammary epithelial cell line as determined by western blot. *, P<0.05. IHC, immunohistochemistry; qRT-PCR, quantitative real-time polymerase chain reaction; TIPE3, tumor necrosis factor-alpha-induced protein 8-like 3.

Transgenic breast cancer cell lines with TIPE3 overexpression and knockdown were constructed

To explore the potential biological function of TIPE3, MCF7 breast cancer cell line with low relative TIPE3 expression and low invasion and migration ability was infected with LV-TIPE3 or blank control LV. MDA-MB-231 breast cancer cell line with high relative TIPE3 expression and high invasion and migration ability was infected with LV-shTIPE3 (1#, 2# or 3#) or LV-shcontrol. As shown in Figure 2A, almost all the LV-infected MCF7 and MDA-MB-231 breast cancer cells expressed GFP, demonstrating extremely high transgenic efficiency. In order to detect the overexpression and knockdown efficiency of LV-mediated TIPE3 and TIPE3 shRNA transduction in MCF7 and MDA-MB-231 breast cancer cells, the transcriptional and protein expression levels of TIPE3 were analyzed by qRT-PCR (Figure 2B) and western blot (Figure 2C,2D). Compared with MCF7-mock control group, mRNA and protein expression levels of TIPE3 in MCF7-TIPE3 breast cancer cells were significantly increased (P<0.05). It was found that compared with MDA-MB-231-shcontrol, the expression mRNA and protein levels of TIPE3 in MDA-MB-231-shTIPE3 1# and MDA-MB-231-shTIPE3 2# in breast cancer cells decreased to different extent, MDA-MB-231-shTIPE3 1# knockdown efficiency was the most significant (P<0.05); however, MDA-MB-231-shTIPE3 3# was almost unchanged. The above results showed that breast cancer transgenic cell lines with stable overexpression and knockdown of TIPE3 were successfully established.

Figure 2 Transgenic breast cancer cell lines with TIPE3 overexpression and knockdown were constructed. (A) MCF7 and MDA-MB-231 transgenic breast cancer cells in bright light and GFP observed by immunofluorescence microscopy. (B) qRT-PCR analysis of relative TIPE3 mRNA expression levels in MCF7 and MDA-MB-231 breast cancer cells, compared with MCF7-mock control group and MDA-MB-231-shcontrol group, respectively. (C) Representative western blot images of TIPE3 protein expression in MCF7 and MDA-MB-231 transgenic breast cancer cells. (D) Western blot analysis of relative TIPE3 protein expression levels in MCF7 and MDA-MB-231 breast cancer cells, compared with MCF7-mock control group and MDA-MB-231-shcontrol group, respectively. *, P<0.05. GFP, green fluorescent protein; qRT-PCR, quantitative real-time polymerase chain reaction; TIPE3, tumor necrosis factor-alpha-induced protein 8-like 3.

TIPE3 can promote the growth and proliferation of breast cancer cells

To investigate the effect of TIPE3 on human breast cancer cells in vitro, MCF7-TIPE3 vs. MCF7-mock and MDA-MB-231-shTIPE3 vs. MDA-MB-231-shcontrol breast cancer cells were detected by CCK-8 cell proliferation assay. It was shown in Figure 3A, overexpressing TIPE3 significantly promoted the growth of breast cancer cells, whereas knockdown TIPE3 inhibited breast cancer cell proliferation (P<0.05). Colony formation assay also confirmed that TIPE3 overexpression breast cancer cells formed larger and more colonies, while the number of colonies of TIPE3 knockdown breast cancer cells was decreased, indicating that TIPE3 enhances the clonogenic ability of breast cancer cells (P<0.05) (Figure 3B,3C). Flow cytometry analysis (Figure 3D) revealed that TIPE3 overexpression decreased the number of cells in G1 phase while increasing the number of cells in S phase, whereas TIPE3 knockdown had the opposite effect. The results indicated that TIPE3 could promote G1-to-S phase transition of breast cancer cells and accelerate the malignant process of cell cycle. To further clarify whether TIPE3 could also promote breast cancer cell growth in vivo, the xenograft model of MDA-MB-231 breast cancer cell in athymic BALB/c nude mice was established. The growth rate of MDA-MB-231-shTIPE3 vs. MDA-MB-231-shcontrol breast cancer cells in BALB/c nude mice was detected and compared by measuring tumor volume and weight (Figure 3E-3G). It was found that the growth of MDA-MB-231-shTIPE3 group was significantly slower than that of MDA-MB-231-shcontrol group (P<0.05). The results demonstrated that TIPE3 could promote the growth and proliferation of breast cancer cells in vitro and in vivo.

Figure 3 TIPE3 can promote the growth and proliferation of breast cancer cells. (A) Results of CCK-8 assay on breast cancer cells. (B) Representative images of breast cancer cell colony formation. The colonies were stained with 0.5% crystal violet. (C) Relative clonogenic ability of MCF7-TIPE3 cells and MDA-MB-231-shTIPE3 cells, compared with MCF7-mock and MDA-MB-231-shcontrol group, respectively. (D) Flow-cytometry analysis was performed to detect cell cycle progression. (E) Tumor volume, compared with the MDA-MB-231-shcontrol group, at 2, 3, and 4 weeks after tumor cell inoculation. (F) Image of xenograft tumors. (G) Tumor weight, compared with MDA-MB-231-shcontrol group. *, P<0.05. CCK-8, Cell Counting Kit-8; TIPE3, tumor necrosis factor-alpha-induced protein 8-like 3.

TIPE3 can promote the migration, invasion and metastasis of breast cancer cells

In order to study the effect of TIPE3 on the migration and invasion of breast cancer cells in vitro, MCF7-TIPE3 vs. MCF7-mock and MDA-MB-231-shTIPE3 vs. MDA-MB-231-shcontrol were compared and analyzed by wound healing assay and transwell invasion assay. As shown in Figure 4A-4D, TIPE3 overexpression promoted the in vitro migratory and invasive ability of MCF7 breast cancer cells, while TIPE3 knockdown inhibited the in vitro migratory and invasive ability of MDA-MB-231 breast cancer cells (P<0.05). Additionally, MDA-MB-231-shTIPE3 vs. MDA-MB-231-shcontrol breast cancer cells were respectively injected into BALB/c nude mice through tail vein to establish the lung metastasis model of breast cancer cells (Figure 4E,4F). The HE staining analysis results showed that TIPE3 knockdown significantly inhibited the lung metastasis ability of MDA-MB-231 breast cancer cells in nude mice (P<0.05). These results indicated that TIPE3 could promote the migration, invasion and metastasis of breast cancer cells in vitro and in vivo.

Figure 4 TIPE3 can promote the migration, invasion and metastasis of breast cancer cells. (A) Representative images of wound healing assay. (B) Relative migratory ability of breast cancer cells: MCF7-TIPE3 vs. MCF7-mock; MDA-MB-231-shTIPE3 vs. MDA-MB-231-shcontrol. (C) Representative images of transwell invasion assay. The invaded cells were stained with 0.5% crystal violet. (D) Relative invasive ability of breast cancer cells: MCF7-TIPE3 vs. MCF7-mock; MDA-MB-231-shTIPE3 vs. MDA-MB-231-shcontrol. (E) Representative images of HE staining in nude mouse lung tissues. (F) The number of metastasis, compared with MDA-MB-231-shcontrol. *, P<0.05. HE, hematoxylin and eosin; TIPE3, tumor necrosis factor-alpha-induced protein 8-like 3.

Mechanism of TIPE3 promoting breast cancer cell growth and metastasis

TIPE3 promoted the activation of AKT, the inactivation of GSK3β, and the up-regulation of β-catenin, Snail1 and Slug in breast cancer cells

A previous study (14) showed that TIPE3, as a novel transporter protein of PIP2 and PIP3 lipid second messengers, is involved in the transport and metabolism of phosphoinositide, and plays an important role in promoting the activation of the PI3K-AKT signaling pathway. Activated AKT can phosphorylate GSK3β to inactivate the kinase activity of GSK3β, which in turn inhibits GSK3β from phosphorylating β-catenin, Snail1, and Slug, and upregulates β-catenin, Snail1, and Slug by blocking their degradation (21-23,44). In order to analyze the molecular mechanism of TIPE3 promoting malignant biological behaviors of breast cancer cell growth, invasion and metastasis, western blot was used to detect the effect of TIPE3 overexpression (MCF7-mock vs. MCF7-TIPE3) and TIPE3 knockdown (MDA-MB-231-shcontrol vs. MDA-MB-231-shTIPE3) on the levels of AKT-GSK3β-β-catenin/Snail axis-related proteins, including p-AKT, AKT, p-GSK3β, GSK3β, β-catenin, Snail1, Slug in breast cancer cells. As shown in Figure 5A, TIPE3 overexpression significantly upregulated the levels of p-AKT, p-GSK3β, β-catenin, Snail1 and Slug in MCF7 breast cancer cells and the levels of nuclear β-catenin, Snail1 and Slug; however, TIPE3 knockdown downregulated the levels of p-AKT, p-GSK3β, β-catenin, Snail1 and Slug in MDA-MB-231 breast cancer cells and the levels of nuclear β-catenin, Snail1 and Slug. Immunohistochemical methods were used to detect the levels of p-AKT, AKT, p-GSK3β, GSK3β, β-catenin, Snail1 and Slug in the subcutaneous xenograft tumor tissues of MDA-MB-231-shcontrol vs. MDA-MB-231-shTIPE3 in nude mice (Figure 5B). It was also confirmed that TIPE3 knockdown could inhibit the expression of p-AKT, p-GSK3β, β-catenin, Snail1 and Slug in MDA-MB-231 breast cancer cells in nude mice. The above results indicate that TIPE3 is likely to be involved in promoting the unregulated growth, invasion, and metastasis of breast cancer cells by up-regulating AKT signaling, inactivating GSK3β, and inhibiting ubiquitinated proteasome-mediated degradation of β-catenin, Snail1, and Slug, and then up-regulating nuclear transcriptional signaling of β-catenin, Snail1, and Slug.

Figure 5 Mechanism of TIPE3 promoting breast cancer cell growth and metastasis. (A) Representative images of AKT-GSK3β-β-catenin/Snail axis-related proteins detected by western blot. (B) Representative images of immunohistochemical analysis of TIPE3 expression of AKT-GSK3β-β-catenin/Snail axis-related proteins in xenograft model of MDA-MB-231 breast cancer cell. (C) Representative images of western blot analysis of cyclin D1, E-cadherin, N-cadherin, vimentin. (D) The relative mRNA expression of cyclin D1, E-cadherin, N-cadherin, vimentin in MCF7-TIPE3 vs. MCF7-mock and MDA-MB-231-shcontrol vs. MDA-MB-231-shTIPE3 detected by qRT-PCR. *, P<0.05. AKT, protein kinase B; GSK3β, glycogen synthase kinase 3β; qRT-PCR, quantitative real-time polymerase chain reaction; TIPE3, tumor necrosis factor-alpha-induced protein 8-like 3.

TIPE3 upregulated the expression of cyclin D1, N-cadherin and vimentin, while downregulated the expression of E-cadherin in breast cancer cells

To further analyze whether TIPE3-induced up-regulation of nuclear transcriptional signals of β-catenin, Snail1, and Slug in breast cancer cells could affect the expression of downstream target genes of β-catenin, Snail1, and Slug in breast cancer cells, including cytokinin cyclin D1, epithelial marker E-cadherin, mesenchymal marker N-cadherin, and vimentin. Meanwhile, western blot (Figure 5C) and qRT-PCR (Figure 5D) were used to detect and compare the levels of cyclin D1, E-cadherin, N-cadherin and vimentin in TIPE3 overexpressed (MCF7-mock vs. MCF7-TIPE3) and TIPE3 knockdown (MDA-MB-231-shcontrol vs. MDA-MB-231-shTIPE3) breast cancer cells. The results also confirmed that TIPE3 significantly upregulated the expression of cyclin D1, N-cadherin and vimentin, while downregulated the expression of E-cadherin in breast cancer cells (P<0.05).

Discussion

Recent evidence (4,14,16,17) has shown that TIPE3, as a novel transporter of lipid second messengers of PIP2 and PIP3, is upregulated in various tumors and plays a critical role in tumor progression and metastasis by promoting the activation of the PI3K-AKT signaling pathway. In this study, breast cancer transgenic cells with TIPE3 overexpression and knockdown were established to perform gain-of-function and loss-of-function assays. The effect and mechanism of TIPE3 on breast cancer were investigated in cell models, animal models and clinical specimen. Our findings indicate that TIPE3 is significantly upregulated in human breast cancer tissues and cells. TIPE3 overexpression promotes breast cancer cell proliferation, G1-to-S phase transition, migration, and invasion, whereas TIPE3 knockdown inhibits cell proliferation, migration, invasion, induces G1 phase arrest, and suppresses tumor growth and lung metastasis in nude mice. Moreover, TIPE3 overexpression markedly upregulates the levels of p-AKT, p-GSK3β, β-catenin, Snail1, Slug, and nuclear β-catenin, Snail1, and Slug in breast cancer cells, along with increased expression of cyclin D1, N-cadherin, and vimentin, while downregulating E-cadherin expression. Conversely, TIPE3 knockdown exerts the opposite regulatory effects in breast cancer cells and subcutaneous xenograft tumors in nude mice.

Abnormal activation of β-catenin signaling is closely associated with various diseases, and plays a critical role in tumor progression and metastasis by transcriptionally activating downstream target genes including c-Myc, cyclin D1, and MMP7 (24-29). GSK3β is a key kinase component of the β-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), GSK3β, casein kinase 1α (CK1α), and its phosphorylation level and activity regulation are also regulated not only by Wnt-dependent pathways but also by various kinases, including AKT (21-23). Upon phosphorylation by AKT, GSK3β is inactivated, thereby preventing the phosphorylation of β-catenin, blocking its proteasomal degradation, and subsequently upregulating β-catenin nuclear transcriptional signaling (22,23). Notably, protein kinase D1 (PKD1) phosphorylates Snail1 at Ser11, promoting its nuclear export and cytoplasmic localization through binding with 14-3-3σ (45). Phosphorylation of Ser residues near the nuclear export sequence (NES) of Snail1 facilitates its cytoplasmic distribution via a chromosome region maintenance 1 (CRM1)-dependent mechanism (46). However, p21-activated kinase 1 (PAK1) and large tumor suppressor kinase 2 (LATS2) is able to promote Snail1 nuclear localization, stability, and transcriptional repression by phosphorylating Snail1 at Ser246 and Thr203, respectively (47,48). Given that TIPE3 promotes PI3K-AKT signaling, we hypothesized that the high expression of TIPE3 in breast cancer may play a crucial role in AKT hyperactivation and subsequent GSK3β inactivation, inhibiting the ubiquitin-proteasomal degradation of β-catenin, Snail1, and Slug, thereby upregulating their nuclear transcriptional signaling and promoting breast cancer progression and metastasis. As expected, western blot and immunofluorescence analysis revealed that TIPE3 overexpression significantly upregulated the levels of p-AKT, p-GSK3β, β-catenin, Snail1, and Slug, as well as their nuclear localization in breast cancer cells, whereas TIPE3 knockdown downregulated these proteins. Furthermore, TIPE3 upregulated the expression of β-catenin, Snail1, and Slug downstream target genes cyclin D1, N-cadherin, and vimentin, while downregulating E-cadherin expression. EMT also plays a pivotal role in tumor cell migration, invasion, and metastasis (30-41). The initiation and activation of EMT are mediated by EMT-inducing transcription factors (EMT-TFs), including Snail1/Slug, Zeb1/2, and Twist1/2, which directly or indirectly suppress the expression of the epithelial marker E-cadherin while activating the expression of mesenchymal markers such as N-cadherin and vimentin (30-41). The expression of EMT-TFs is regulated at multiple levels, including transcription, post-transcription, translation and post-translation (33-37,40,41). Our findings suggest that TIPE3 may promote breast cancer cell growth, EMT, and metastasis by regulating the AKT-GSK3β-β-catenin/Snail axis and enhancing β-catenin, Snail1, and Slug signaling.

Although this study systematically investigated the role and potential mechanisms of TIPE3 in breast cancer progression across cellular, animal, and clinical specimen models, several limitations should be acknowledged. First, the functional experiments were primarily conducted in a single breast cancer cell line, and the biological effects and regulatory mechanisms of TIPE3 have not been comprehensively validated across different molecular subtypes of breast cancer, such as HER2-positive and triple-negative subtypes, which may exhibit distinct responses. Second, the proposed AKT-GSK3β-β-catenin/Snail axis remains correlative in nature, and further studies employing kinase inhibitors, E3 ligase interference, or other molecular tools are required to determine whether TIPE3 modulates this pathway directly or indirectly. In addition, although we preliminarily assessed TIPE3 expression in clinical breast cancer specimens and its association with certain clinicopathological features, the limited sample size precluded robust statistical validation, including survival analysis, subtype stratification, and multivariate regression, making it difficult to determine whether TIPE3 serves as an independent prognostic biomarker.

Conclusions

In conclusion, TIPE3 represents a newly identified regulatory factor in breast cancer that may promote tumor proliferation, EMT, and distant metastasis through activation of the AKT-GSK3β-β-catenin/Snail signaling axis. Future studies should aim to expand validation across molecular subtypes, incorporate lipid signaling tools, and analyze larger clinical cohorts to further elucidate its functional network and clinical relevance, thereby providing new theoretical insights and potential targets for personalized breast cancer therapy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-717/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-717/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-717/prf

Funding: This work was supported by a research grant from the National Natural Science Foundation of China (No. 82260551), the Project Funding from Suzhou Ninth People’s Hospital (No. YK202320), and Project in Science and Education of Wujiang District (No. wwk202317).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-717/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All participating patients signed an informed consent form, and the study was approved by the Medical Ethics Committee of The First Affiliated Hospital of Soochow University (IRB No. 2021-036). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Animal experiments were performed under a project license (IRB No. A201809059) granted by Laboratory Animal Center of Soochow University, in compliance with its institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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