AXL is associated with STAT3 activation in breast cancer

Introduction

Breast cancer (BC) is the most common malignancy affecting women worldwide, with diverse molecular subtypes and complex pathogenic mechanisms (1-3). In 2022, it was estimated that there were >2 million new BC cases worldwide, accounting for 11.6% of all cancer cases. A subset of BCs, known as triple-negative BC (TNBC), lacks the expression of estrogen receptor (ER), progesterone receptor (PR) and HER2, and accounts for ~10-20% of all BC cases. TNBC often belongs to the basal-like molecular subtype. These tumors are aggressive with a poor prognosis due to ineffective target therapies. Therefore, identifying key signaling pathways driving BC progression is crucial for developing effective therapeutic strategies (4,5).

AXL is a receptor tyrosine kinase of the TAM (Tyro3, AXL, Mer) family, activated upon binding its ligand growth arrest-specific 6 (Gas6) (6-9). AXL is frequently overexpressed in BC, particularly TNBC, and promotes epithelial-to-mesenchymal transition and metastasis and is associated with a poor prognosis by activating multiple downstream signaling pathways, including phosphoinositide 3-kinase (PI3K) and AKT (protein kinase B), as well as mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) (10-15).

Signal transducer and activator of transcription 3 (STAT3) is a critical transcription factor that mediates signaling from cytokines (for example, IL-6) and growth factors (16,17). The aberrant activation of STAT3, primarily via tyrosine-705 phosphorylation, is a common oncogenic event in BC (18). Activated STAT3 dimerizes and translocates to the nucleus to regulate the transcription of genes governing cell cycle progression, anti-apoptosis and metastasis. The IL-6/JAK/STAT3 signaling axis is well-documented as a driver of BC progression and drug resistance (19,20).

Emerging evidence suggests a crosstalk between AXL and STAT3 signaling in multiple cancer types. A previous study by the authors revealed an association between AXL and STAT3 activation in esophageal squamous cell carcinoma by Gene Set Enrichment Analysis (GSEA) analysis (21). Khera et al (22) reported that the activation of AXL by GAS6 induced the phosphorylation of proline-rich tyrosine kinase 2, a non-receptor tyrosine kinase that serves as an essential upstream mediator of STAT3 activation. In a previous study on gastric cancer, co-culture with cancer-associated fibroblasts (CAFs) triggered STAT3 activation, which was reversed by the AXL inhibitor, 9im, although the selectivity of 9im requires further validation (23). Similarly, Hung et al (24) demonstrated that AXL signaling within lung cancer cells and CAF directly drives the secretion of IL-11, which binds to the GP130 complex and activates STAT3 in tumor-associated macrophages (TAMs). Concordant AXL-STAT3 signaling polarizes TAMs toward an M2-like phenotype, upregulates CD44 and mesenchymal markers, and enhances macrophage-mediated vasculogenic network formation. The dual inhibition of AXL and STAT3 disrupts this paracrine loop, reduces TAM recruitment and polarization, and suppresses tumor growth in lung cancer xenograft models (24). However, the direct functional link between AXL and STAT3 activation in BC remains poorly defined.

Thus, it was hypothesized that AXL modulates STAT3 activation in BC cells. Using in vitro experiments and clinical dataset analyses, the present study aimed to validate this hypothesis and to establish a functional association between AXL and STAT3 signaling in BC.

Materials and methods

Cell lines and reagents

MCF7 (cat. no. SCSP-531), 293T (cat. no. SCSP-502), MDA-MB-231 (cat. no. SCSP-5043) and HeLa (cat. no. SCSP-504) cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. MCF7 cells were cultured in MEM (Procell Life Science & Technology, Co., Ltd.) supplemented with 0.01 mg/ml insulin and 10% fetal bovine serum (FBS). The 293T, MDA-MB-231 and HeLa cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS. All cells were incubated at 37˚C in a humidified atmosphere with 5% CO2. Cell line authentication was performed by short tandem repeat profiling at the Forensic Science Center, Jining Medical University. IL-6 was purchased from PeproTech, Inc. R428 and tocilizumab were obtained from MedChemExpress. R428 was used as previously described (25), and tocilizumab was used at 25 µg/ml.

Plasmids, short hairpin RNA (shRNA) and transfection

AXL plasmid (cat. no. 105932, based on pcDNA4/TO/myc/HIS) was obtained from Addgene, Inc. The STAT3 reporter plasmid (SIE vector) and pRL-TK vector were purchased from Promega Corporation. Lentivirus for shRNA targeting AXL (shAXL: 5'-GGGTGACAATGTGGGAGATTG-3') and control shRNA (Ctrl: 5'-TTCTCCGACGTGTCACGT-3') were purchased from Shanghai GenePharma Co., Ltd. as pre-packaged, ready-to-use viral particles (3rd-generation, packaged in 293T cells; titer 109 TU/ml). Transfection of plasmids (1 µg for one well of 12-well plate) was performed using Lipo8000 (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, Lipo8000 and plasmids were incubated in DMEM without FBS for 5 min and added the cell medium for 4 h at 37˚C. A total of 44-48 h later, cells were harvested for analysis. For lentiviral transfection, viruses (5 µl/ml) and polybrene (2 µg/ml) were added to the cell medium at MOI=5, incubated at 37˚C/5% CO2 for 16 h before medium change. After 3 days cells were treated with puromycin (2 µg/ml) for 1 week to select transfected cells. Then, cells were cultured at puromycin (0.5 µg/ml) for maintenance. These stable cell lines were used for experiments.

Nuclear and cytoplasmic protein extraction

Nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic protein extraction kit (cat. no. P0027; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. These experiments were repeated three times.

Western blot analysis

Western blot analysis was performed as previously reported (25,26). Total cellular proteins were extracted using RIPA buffer supplemented with protease and phosphatase inhibitors sourced from Beyotime Institute of Biotechnology. Subsequently, nuclear and cytoplasmic proteins were isolated using a dedicated nuclear-cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology). The protein concentration was precisely measured using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal quantities (10 µg) of these proteins were resolved through 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were first blocked for 1 h at room temperature in a 5% non-fat milk solution. They were then incubated overnight at 4˚C with an array of primary antibodies: AXL (1:1,000; cat. no. 8661), phosphorylated (p)-AKT (1:1,000; cat. no. 4060), STAT3 (1:1,000; cat. no. 9139), Histone H3 (1:1,000; cat. no. 9715), p-STAT3 (1:1,000; cat. no. 9145), AKT (1:1,000; cat. no. 4691; Cell Signaling Technology, Inc.), ERK1 + ERK2 (1:10,000; cat. no. ab184699), p-ERK1 + ERK2 (1:10,000; cat. no. ab76299) (all from Abcam), GAPDH antibody (1:1,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.) and p-AXL (1:200; cat. no. AF2228; R&D Systems, Inc.). Post-incubation with the primary antibodies, the membranes were further incubated with peroxidase-conjugated goat anti-rabbit (cat. no. ZB-2301) or goat anti-mouse (cat. no. ZB-2305) secondary antibodies, both from OriGene Technologies Inc., at a dilution of 1:5,000 for 1 h at room temperature. Immunoreactive bands were detected using ECL reagent from Beyotime Institute of Biotechnology. GAPDH and histone H3 were employed as loading controls for cytoplasmic and nuclear proteins, respectively. All the experiments were repeated at least three times. ImageJ software (version 1.53m; National Institutes of Health) was used for the densitometric analysis.

Dual luciferase reporter gene assay

Cells underwent co-transfection with an SIE vector serving as a STAT3 reporter, a pRL-TK vector acting as an internal control, and either an empty vector (EV) or an AXL plasmid using Lipo8000 (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. For each well of a 12-well plate, a total of 1 µg plasmid DNA was transfected, comprising 0.5 µg AXL plasmid (or empty vector), 0.45 µg STAT3 reporter plasmid (SIE vector), and 0.05 µg pRL-TK internal control plasmid. The transfection complexes were incubated with cells for 4 h at 37˚C in a humidified incubator with 5% CO2, after which the medium was replaced with fresh complete medium carefully. At 48 h post-transfection, luciferase activity was determined by employing the Dual Luciferase Reporter Gene Assay kit from Beyotime Institute of Biotechnology (cat. no. RG029M), strictly adhering to the manufacturer's guidelines. In the inhibitor experiments, cells were transfected with 0.45 µg STAT3 reporter plasmid (SIE vector), and 0.05 µg pRL-TK internal control plasmid. 24 h later, the cells were incubated with R428 at a concentration of 1, 3 µM or DMSO for another 24 h followed by luciferase activity determination. This experiment was repeated three times with triplicate wells for each condition, and data was normalized by comparison with Renilla luciferase activity.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized using PrimeScript RT Master Mix (Takara Biotechnology, Co., Ltd.) according to the manufacturer's instructions. qPCR was performed using SYBR-Green PCR Master Mix (Vazyme Biotech Co., Ltd.) on a QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific, Inc.). Each qPCR reaction was performed in a final volume of 20 µl, containing 10 µl SYBR Green Master mix (2X), 0.5 µl forward and reverse primers (10 µM), 8 µl DEPC treated water and 1 µl cDNA. The following thermocycling conditions were used for the qPCR: 95˚C for 30 sec, 40 cycles of 95˚C for 10 sec and 60˚C for 30 sec. The primer sequences for IL-6 and 18S were as follows: IL-6 forward, 5'-CACAGACAGCCACTCACCTC-3' (sense) and reverse, 5'-TTTTCTGCCAGTGCCTCTTT-3' (antisense); 18S rRNA, 5'-GAGGATGAGGTGGAACGTGT-3' (sense) and reverse, 5'-GGACCTGGCTGTATTTTCCA-3' (antisense). 18S was used as an internal control. Relative gene expression was calculated using the 2-ΔΔCq method (27). This experiment was repeated three times with triplicate wells for each condition.

ELISA

The IL-6 concentration in the cell culture supernatants was measured using an IL-6 ELISA kit [cat. no. EK106; Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.] according to the manufacturer's protocol. This experiment was repeated three times with triplicate wells for each condition.

Bioinformatics analysis

Gene expression datasets GSE102484 (n=683) (28) and GSE9893 (n=155) (29) were downloaded from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/). GSEA was performed to compare IL6_JAK_STAT3 signaling between the AXL-high and AXL-low groups as reported in a previous study by the authors (21). Protein expression data of AXL and p-STAT3 (Y705) from breast invasive carcinoma were obtained from The Cancer Proteome Atlas (TCPA, n=901) (https://www.tcpaportal.org/tcpa/download.html); the samples whose AXL expression marked with NA (n=156) were excluded. The association between the expression of AXL and p-STAT3 was analyzed with SPSS (version 13.0; SPSS, Inc.) using Pearson's correlation analysis (n=745). The association analysis between AXL mRNA and IL-6 mRNA in BC was performed by gepia2 using Pearson's correlation analysis as the method to calculate the correlation coefficient (http://gepia2.cancer-pku.cn/#correlation) (30).

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Differences between two groups were analyzed using a two-tailed unpaired Student's t-test. Comparisons among multiple groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test using GraphPad Prism 8 (GraphPad Software, Inc.; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Results

AXL overexpression enhances STAT3 activation in BC cells

To investigate the effects of AXL on STAT3 activation, AXL was overexpressed in MCF7 and 293T cells. Western blot analysis revealed that AXL overexpression significantly increased p-STAT3 (Y705) levels without altering total STAT3 (t-STAT3) expression (Fig. 1A and B). Moreover, AXL overexpression enhanced p-STAT3 (Y705) levels in both cell lines in a concentration-dependent manner (0, 1 and 2 µg) (Fig. 1C and D).

Figure 1 AXL overexpression enhances STAT3 activation. (A) MCF7 cells were transfected with empty plasmid (EV) or AXL plasmid; the indicated proteins were examined using western blot analysis. (B) 293T cells were transfected with empty plasmid (EV) or AXL plasmid for 24 h and treated with or without IL-6 (10 ng/ml) for 30 min; the indicated proteins were examined using western blot analysis. (C) MCF7 cells were transfected with increasing amounts of AXL plasmid for 48 h and the indicated proteins were examined using western blot analysis. (D) 293T cells were transfected with increasing amounts of AXL plasmid for 48 h and the indicated proteins were examined using western blot analysis. (E) MCF7 cells transfected with empty plasmid (EV) or AXL plasmid; the indicated proteins were examined using western blot analysis. (F) MCF7 cells were co-transfected with empty plasmid (EV) or AXL plasmid, as well as STAT3 reporter plasmid (SIE vector) and pRL-TK vector for 48 h and analyzed using the Dual Luciferase Reporter Gene Assay kit. (G) 293T cells were co-transfected with empty plasmid (EV) or AXL plasmid, as well as STAT3 reporter plasmid (SIE vector) and pRL-TK vector for 48 h and analyzed using the Dual Luciferase Reporter Gene Assay kit. (H) MCF7 cells transfected with empty plasmid (EV) or AXL plasmid; the mRNA expression of IL-6 was determined using reverse transcription-quantitative PCR. (I) MCF7 cells transfected with empty plasmid (EV) or AXL plasmid; IL-6 levels in the cell supernatants was determined using ELISA. (J) Scatter plot illustrating the correlation between the mRNA expression levels of AXL and IL-6 analyzed using the GEPIA2 online tool. (K) MCF7 cells were transfected with EV (-) or AXL plasmid (+) for 48 h and then treated with or without tocilizimab (25 µg/ml) for 24 h. The indicated proteins were examined using western blot analysis. t-, total. *P<0.05, **P<0.01 and ***P<0.001. EV, empty vector; p-, phosphorylated.

Subcellular fractionation analysis revealed that AXL overexpression increased nuclear STAT3 levels in MCF7 cells, indicating an enhanced STAT3 nuclear translocation (Fig. 1E). A port of AXL was also detected in the nucleus, consistent with a previous study (31). Dual luciferase reporter assay confirmed that AXL overexpression significantly augmented STAT3 transcriptional activity in MCF7 and 293T cells (Fig. 1F and G). Additionally, AXL overexpression upregulated IL-6 mRNA expression and secretion, as measured using RT-qPCR and ELISA, respectively (Fig. 1H and I). Furthermore, GEPIA2 analysis further validated an association between AXL and IL-6 mRNA in clinical BC samples (P=4.1x10-7) (Fig. 1J). Treatment with the IL-6 receptor-neutralizing antibody, tocilizumab, partly abrogated the AXL-induced activation of STAT3 (Fig. 1K). These results suggest that AXL activates STAT3, at least in part by promoting IL-6 secretion.

AXL suppression inhibits STAT3 activation

To further confirm the role of AXL in STAT3 activation, AXL was knocked down in MDA-MB-231 and HeLa cells, which express high endogenous AXL. The results of western blot analysis demonstrated that the shRNA-mediated AXL knockdown significantly reduced STAT3 activation (Fig. 2A and B). Pharmacological inhibition with the selective AXL inhibitor, R428, also decreased AXL and STAT3 activation in the MDA-MB-231 cells in a concentration-dependent manner (Fig. 2C and D). Furthermore, R428 treatment dose reduced STAT3 transcriptional activity in a concentration-dependent manner (Fig. 2E).

Figure 2 AXL suppression decreases STAT3 activation. (A) MDA-MB-231 cells were transfected with AXL-shRNA lentivirus (shAXL) or control lentivirus (Ctrl) for 48 h and treated with puromycin (2 µg/ml) for ~1 week to select transfected cells. The indicated proteins were examined using western blot analysis. (B) HeLa cells were transfected with AXL-shRNA lentivirus (shAXL) or control lentivirus (Ctrl) for 48 h and treated with puromycin (2 µg/ml) for ~1 week to select transfected cells. The indicated proteins were examined using western blot analysis. (C and D) MDA-MB-231 cells were treated with increasing concentration of R428 for 24 h. The indicated proteins were examined using western blot analysis. (E) MDA-MB-231 cells were co-transfected with empty plasmid (EV) or AXL plasmid, as well as STAT3 reporter plasmid (SIE vector) and pRL-TK vector for 24 h and treated with DMSO (Ctrl) or R428 (1 and 3 µM) for a further 24 h; STAT3 activity was analyzed using the Dual Luciferase Reporter Gene Assay kit. *P<0.05, **P<0.01, ***P<0.001. shRNA, short-hairpin RNA; EV, empty vector; p-, phosphorylated.

AXL expression correlates with STAT3 signaling in clinical BC samples

GSEA analysis of the GSE102484 and GSE9893 datasets revealed that IL6_JAK_STAT3 signaling was significantly enriched in AXL-high BC tumors compared with AXL-low tumors [normalized enrichment score (NES)=1.57, P=0.027 for GSE102484; NES=1.58, P=0.008 for GSE9893] (Fig. 3A and B). TCPA dataset analysis also revealed a positive correlation between AXL protein expression and p-STAT3 (Y705) levels (Fig. 3C). These clinical data support a close association between AXL and STAT3 activation in BC.

Figure 3 AXL is associated with STAT3 signaling, as revealed by the analysis of mRNA and protein data of clinical breast cancer samples. (A) Gene set enrichment analysis of GSE102484, mRNA expression data from 683 patients with invasive breast cancer; IL6_JAK_STAT3 signaling in AXL-high tumors vs. AXL-low tumors is displayed. (B) Gene set enrichment analysis of GSE9893, mRNA expression data from 155 primary breast cancers; IL6_JAK_STAT3 signaling in AXL-high tumors vs. AXL-low tumors is displayed. (C) Association of AXL and p-STAT3 (pY705) in breast invasive carcinoma; data are from The Cancer Proteome Atlas and were analyzed using Prism 8. NES, normalized enrichment score.

Discussion

The present study demonstrated that AXL was associated with STAT3 activation in BC. AXL overexpression enhanced STAT3 phosphorylation, nuclear translocation and transcriptional activity, whereas AXL knockdown or inhibition suppressed STAT3 activation. Clinical dataset analyses further validated the positive correlation between AXL and STAT3 signaling in samples from patients with BC.

AXL has been reported to promote cancer progression through multiple signaling pathways (6,7,13). The present study identified STAT3 as a novel downstream target of AXL in BC. AXL overexpression increased IL-6 secretion, which may contribute to STAT3 activation via the IL-6/JAK/STAT3 pathway. Additionally, AXL may activate STAT3 through other mechanisms, such as Src and JAK kinases, which are well-characterized upstream activators of STAT3 (32,33). Notably, AXL has been reported to activate Src in multiple cancer types (34-36). However, whether AXL directly phosphorylates STAT3 warrants further investigations.

Constitutive STAT3 activation is a key driver of BC progression, regulating cell proliferation, survival and metastasis (18,19,37). The clinical relevance of the AXL-STAT3 axis is highlighted by their positive correlation in datasets of patients with BC. AXL-high tumors exhibit enhanced STAT3 signaling, which may predict a poor prognosis and therapeutic response. Therefore, the AXL-STAT3 axis represents a potential prognostic biomarker and therapeutic target for BC, particular TNBC, which frequently exhibits the co-overexpression of AXL and STAT3 (19,37-39). From a translational perspective, the AXL-STAT3 signaling axis, as proposed in the present study, represents a subtype-specific actionable target, particularly for TNBC, which lacks effective targeted therapies. A high expression of AXL accompanied by STAT3 hyperactivation may serve as a prognostic biomarker and predictive indicator of poor outcomes and therapeutic resistance in BC, particularly TNBC. The selective AXL inhibitor, R428 (bemcentinib), has exhibited promising antitumor efficacy in preclinical models (11,40-43) and under early-phase clinical trials for TNBC (NCT03184558) (14). The combined inhibition of AXL and STAT3 may further enhance therapeutic efficacy by blocking this oncogenic signaling cascade more completely, providing a novel precision strategy for TNBC and other AXL/STAT3-activated BC subtypes.

The present study has certain limitations that should be acknowledged. First, in vivo experiments to validate the AXL-STAT3 axis are currently lacking, which the authors plan to address in BC animal models in future research. Second, the exact mechanisms by which AXL activates STAT3 require further investigation. In particular, co-immunoprecipitation (co-IP) assays could provide direct biochemical evidence of protein-protein interaction and represent a promising approach to determine whether AXL directly activates STAT3. Finally, future studies are warranted to explore the therapeutic potential of combining AXL and STAT3 inhibition for BC treatment. In conclusion, the present study reveals that AXL is functionally associated with STAT3 activation in BC, providing novel insight into the molecular mechanisms of BC progression. Targeting the AXL-STAT3 axis may represent a promising therapeutic strategy for patients with BC.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by Jining Key Research and Development Plan Project (grant no. 2025YXNS067).

Availability of data and materials

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

Authors' contributions

HZ and QG designed the experiments. GZ, XS, JZ, ZZ, XL, FY, ZG, CG, YaH, MZ, JE, YX, JuL, YuH, XM, RY and JiL performed the experiments. GZ, XS analyzed the data and wrote the manuscript. HZ and QG confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References