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. 2025 Mar 24;16(6):2651–2662. doi: 10.1039/d4md00926f

An allosteric inhibitor targeting the STAT3 coiled-coil domain selectively suppresses proliferation of breast cancer†

Min Huang a,‡,✉, Wei Wang b,‡, Liyue Cao a, Jiaxin Liu a, Can Du b, Jian Zhang c,✉
PMCID: PMC11969721  PMID: 40190419

Abstract

Signal transducer and activator of transcription 3 (STAT3) remains a challenging and attractive therapeutic target in cancer research. The coiled-coil domain (CCD) of STAT3 represents a novel site for targeted intervention, distinct from the Src-homology 2 domain, and plays a crucial role in regulating the earlier activation and biological function of STAT3 in cell proliferation, survival and invasion of breast cancer cells. We previously reported K116, N′-(1-(2,4-dihydroxyphenyl)ethylidene)thiophene-2-carbohydrazide, as a potent allosteric inhibitor specifically targeting the STAT3 CCD. This study aimed to investigate the antiproliferation effect of K116 on breast cancer cells in vitro and in vivo. The results showed that K116 inhibited the proliferation of breast cancer cell lines in a dose-dependent manner by reducing the phosphorylation of STAT3 Lyr705 and did not inhibit the proliferation of HGC-27 and A549 cells nor their STAT3 Lyr705 phosphorylation. Compared with Stattic (STAT3 SH2 inhibitor), K116 selectively inhibited the proliferation of breast cancer cells. Furthermore, K116 (20 μM) directly monitored STAT3 stabilization and engagement within MDA-MB-468 cells, without affecting STAT1, STAT5, and Akt1. K116 induced apoptosis and inhibited migration as well as pY705STAT3 nuclear translocation and transcriptional activity of STAT3. In addition, K116 (30 mg kg−1) markedly suppressed tumor growth and inhibited STAT3 activity in a 4T1 cell-derived murine breast cancer model. Overall, our results provided pharmacological evidence supporting future clinical investigation of K116 as a promising STAT3 CCD allosteric inhibitor for breast cancer treatment.

The proposed mechanism of K116 inhibiting breast cancer through inhibition of the STAT3 signaling pathway.graphic file with name d4md00926f-ga.jpg

1. Introduction

Breast cancer is the most prevalent form of cancer globally, which is usually diagnosed in middle-aged women; the high frequency of metastasis contributes to the extremely poor prognosis.1 Therefore, identifying the molecular pathogenesis underlying breast cancer is critical so that new strategies for treating it can be developed. A large body of cumulative evidence supports signal transducer and activator of transcription 3 (STAT3) as a promising therapeutic target in breast cancer.2

STAT3 is an early tumor diagnostic marker known to promote breast cancer malignancy.3 Accumulating evidence indicates the involvement of overexpressed and constitutively activated STAT3 in the progression, proliferation, metastasis, and chemoresistance of breast cancer.4 STAT3 dimerization occurs through the interaction of phosphopeptide-containing pTyr705 from one monomer with the binding pocket in the Src-homology 2 (SH2) domain of another monomer.5,6 The screening of small molecules that inhibit STAT3 activity by disrupting the SH2 domain has been emphasized in the last two decades, for example, Stattic,7–9 STA-21, and B9. Unfortunately, these diverse sets of compounds are used to optimize leads and clinical development. Consequently, many STAT3 inhibitors are in preclinical development or under evaluation in clinical trials for their therapeutic efficacy predominantly in cancer.10,11 Indeed, it is difficult to discover small molecules that bind to the STAT3 SH2 domain with high affinities and sufficient selectivity due to the high sequence homology of the SH2 domain in many proteins in cells.12 This may lead to a lack of specificity and pharmacological efficacy. Therefore, interfering with other domains of STAT3 is important in designing drugs targeting STAT3.

The coiled-coil domain (CCD) of STAT3 has been recognized as a crucial region for nearly 20 years, with a well-defined functional role reported.13 The STAT CCD comprises a four-helix structure consisting of approximately 194 amino acids, occupying roughly one-fourth of the protein and playing an essential role in recruiting STAT3 to both cytokines and growth factor receptors. This recruitment leads to subsequent SH2 Tyr705 phosphorylation, dimer formation, nuclear translocation, and DNA binding.14,15 Studies have revealed the mechanism for SH2 regulation via the CCD and have shown that allosteric regulation of SH2 via the coiled-coil domain (CCD) is an alternative drug design strategy. The allosteric mechanism provides new insight into the understanding of intramolecular signaling in STAT3 and potential pharmaceutical control of STAT3 specificity and activity. Thus, targeting the CCD is desired for a downstream effect on the SH2 domain and the overall STAT3 function. However, the full exploration of the anticancer efficacy of the STAT3 CCD in breast cancer is hindered by the lack of cellular CCD inhibitors.16–18

Our previous study identified K116 as a novel allosteric inhibitor of the STAT3 CCD.19 Nevertheless, whether the allosteric inhibitor K116 is selectively efficient for breast cancer cells and holds promise as a therapeutic strategy for breast cancer remains unclear. This study provided further evidence demonstrating the valuable therapeutic efficacy of K116 in both in vitro and in vivo models of breast cancer.

2. Materials and methods

2.1. Synthesis of K116, K134 and K114

K116, (E)-N′-(1-(2,4-dihydroxyphenyl)ethylidene)thiophene-2-carbohydrazide, K134, (E)-N′-(1-(2,4-dihydroxyphenyl)ethylidene)-2-(o-tolyloxy)acetohydrazide, and K114, (E)-N′-(1-(2,4-dihydroxyphenyl)ethylidene)-2-hydroxybenzohydrazide. All target compounds were confirmed to have >95% purity. The synthesis procedures of K116, K134 and K114 are provided in the ESI.†

2.2. Cell culture

The MDA-MB-468 (human breast cancer cells), 4T1 (mouse breast cancer cells), MCF-7 (human breast cancer cells with normal pY705STAT3), Huh-7 (human hepatocarcinoma cells), SMMC-7721 (human hepatocarcinoma cells), BEL-7404 (human hepatoma cells), MKN28 (human gastric cancer cells), MKN45 (human gastric cancer cells), HGC-27 (human gastric cancer cells), A549 (human non-small-cell lung cancer cells), 293T (human embryonic kidney cells), and AC16 (human hybrid cells) cell lines were obtained from the Cell Resource Center of the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, and Procell Life Science & Technology Co. Ltd. The MDA-MB-468 cells were maintained in L15 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, USA). The MCF-7, 4T1, Huh7, SMMC-7721, BEL-7404, MKN28, MKN45, and HGC27 cells were maintained in DMEM supplemented with 10% (v/v) FBS (Invitrogen). The A549 cells were maintained in F-12K medium supplemented with 10% (v/v) FBS. The MDA-MB-468 cells were cultured following the standard cell-culturing protocols of the American Type Culture Collection and maintained in a humidified 37 °C incubator with 5% CO2. The other cell lines were also cultured following the standard cell-culturing protocols of the American Type Culture Collection and maintained in a humidified 37 °C incubator with 5% CO2.

2.3. Cell viability

The cell viability was evaluated using a cell counting kit-8 (CCK-8) (Biotech and Dojindo) following the manufacturer's protocols. Briefly, the cells were seeded in 96-well plates at a density of 5 × 103 cells per well. After attaching overnight, the cells were treated with various concentrations of K116. The cells treated with DMSO were used as vehicles (negative control). After 24 h of treatment, CCK-8 (10 μL per well) was added and incubated at 37 °C for 4 h. The absorbance of each well was detected at 450 nm using a microplate reader (Synergy H4 Hybrid Reader, BioTek, USA) to measure the number of viable cells. The IC50 values were calculated using GraphPad Prism 10. Each data point represented the mean ± standard error of the mean (SEMs) of three independent experiments.

2.4. Immunoblotting

The cells (3 × 105 to 5 × 105 cells) were seeded in six-well plates and treated with DMSO or K116. After 24 h of treatment, both floating and adherent cells were lysed with 1× sodium dodecyl sulfate (SDS) buffer, separated using SDS–polyacrylamide gel electrophoresis (PAGE), and transferred onto a nitrocellulose (NC) membrane. After blocking with 5% skimmed milk, the cells were incubated with specific primary antibodies (Table S4†) overnight at 4 °C and then with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G. An Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, USA) was used for detection.

2.5. Cellular thermal shift assay

The MDA-MB-468 cells were incubated with or without K116 for 24 h and washed with phosphate-buffered saline (PBS). Two groups, the K116 group and the vehicle group (DMSO), were diluted in PBS. All buffers were supplemented with complete protease inhibitor cocktail. The cells were divided into smaller aliquots (80 μL) and heated individually at different temperatures (34 °C, 39 °C, 44 °C, 49 °C, 54 °C, and 59 °C) for 3 min (Life Technologies, USA), followed by cooling at 4 °C for 3 min. The cell suspensions were freeze-thawed three times using liquid nitrogen. The soluble fraction (lysate) was separated from the cell debris by centrifugation at 20 000g and 4 °C for 30 min. The supernatants were transferred to new microtubes and analyzed using SDS-PAGE followed by western blot analysis (STAT3, STAT1, STAT5, and Akt).20 For the cellular thermal shift assay (CETSA) curves, the band intensities were related to the intensities of the lowest temperature for the drug-exposed and vehicle samples. The band intensities were analyzed using ImageJ software. The data (band intensities) were plotted using GraphPad Prism 10 software.

2.6. Luciferase reporter

TargetScan (https://www.targetscan.org) was used to predict the binding site of STAT3. The recombinant reporter plasmid pmir-STAT3 3′-UTR was constructed and transfected into HeLa cells using Lipofectamine 2000. The luciferase reporter activity was evaluated after 24 h of treatment with increasing concentrations (6.25 μM, 12.5 μM and 25 μM) of K116. The relative luciferase values were the ratio of the absolute activity of firefly luciferase to that of Renilla luciferase.21 The results of the luciferase assay represented the averages from three independent experiments.

2.7. Flow cytometry

The 4T1 cells (5 × 105 cells) were seeded in six-well plates. After 24 h of seeding, the cells were treated with DMSO and K116 for 24 h. Then, both floating and adherent cells were collected and fixed with 75% ethanol at 4 °C overnight. The cells were incubated with RNase I (50 μg mL−1) in 1× PBS at 37 °C for 30 min and stained with propidium iodide (PI) (50 μg mL−1) for 15 min. The cells were fixed with 70% ice-cold ethanol, washed with PBS, incubated with RNase (50 μg, 10 109 134 001, Sigma-Aldrich, USA) at 37 °C for 1 h, and stained with PI (20 μg, 556 463; BD Biosciences, CA, USA) at 4 °C in the dark to analyze apoptotic cell population. Next, Annexin V binding buffer (422 201; BioLegend, CA, USA) containing fluorescein isothiocyanate conjugated with an anti-Annexin V antibody (640 906, 1, 50 dilution; BioLegend) was used for Annexin V staining following the manufacturer's protocol. The stained cells were assessed using flow cytometry and FACS DIVA software, v6.2 (BD Biosciences). The cell cycle distribution was analyzed using FlowJo 7.6.1 software.

2.8. Wound-healing assay

The cell migration was assessed using a wound-healing assay. The 4T1 cells (1 × 105 cells) were seeded in 24-well plates with 10% FBS-containing media and cultured until they reached 90% confluence. The wound was introduced by scraping the cell monolayer with a sterile 200 μL pipette tip, and the cells were treated with indicated concentrations of K116. After 0 and 24 h, the wound areas were quantified by measuring the width of the cell-free zone at six distinct positions with a digitally calibrated micrometer using microphotographs at 10× magnification, and the images were taken with an Olympus inverted microscope equipped with a CCD camera.

2.9. Cell invasion assays

K116 was mixed with or without MDA-MB-468 and 4T1 cells at the indicated concentrations (1 μM and 10 μM) for 24 h. We performed cell invasion assays using Matrigel matrix-coated Transwell chambers (Costar Corning, MA, USA). The MDA-MB-468 cells were seeded into the upper wells at a density of 8.5 × 105 cells per well (200 μL). L15 and DME media (800 μL) containing 10% FBS were added to the bottom wells of the plate. The membranes were washed with PBS. The noninvaded cells were scraped off, and the invaded cells on the bottom of the Transwell membrane were immobilized with 4% paraformaldehyde and stained with crystal violet. The stained cells were visualized and counted from five randomly selected fields using an inverted fluorescence microscope (Olympus Microsystems). Directional migration was quantified by cell counting using ImageJ software (National Institutes of Health, MA, USA).

2.10. Immunofluorescence assay

The MDA-MB-468 cells were grown on coverslips. The coverslips were washed twice with PBS at 25 °C, fixed in 4% paraformaldehyde at room temperature for 15 min, washed with 1× PBS twice, fixed with methanol for 30 min at room temperature, and incubated in blocking solution (PBS containing 5% bovine serum albumin) for 1 h at room temperature. After blocking, the cells were incubated in blocking solution containing approximately 2 μg mL−1 of each primary antibody (pY705STAT3; Cell Signaling Technology) overnight at 4 °C. The cells were then washed three times with PBS and incubated in blocking solution containing secondary antibodies for 1 h at room temperature. After incubation, the cells were washed three times with PBS, DNA stained with 1 μg mL−1 4′,6-diamidino-2-phenylindole (DAPI) for 3 min on the shaker, washed with PBS, and mounted. The cells were imaged on an inverted fluorescence microscope (Olympus, Japan).

2.11. Studies using a 4T1 cell-derived murine breast cancer model

Briefly, 4T1 cells (1 × 106) suspended in 100 μL of 1× PBS were subcutaneously implanted into the right galactophore of 14 C57BL/6 female mice (6–8 weeks, SPF) to establish the xenograft tumor model. The mice were monitored daily, and caliper measurements started when the tumors became visible. The tumor volumes were measured every other day using calipers. Then, the tumor volumes were calculated using the following formula: (L × W2)/2, where L and W refer to the length and width of tumors, respectively. When the tumor size reached approximately 100–300 mm3, the mice were randomized into two groups (n = 6 per group): (a) vehicle and (b) 30 mg kg−1 K116. The mice received an intraperitoneal injection of the vehicle alone (1% DMSO, 5% polyoxyethylene castor oil, and 94% saline) or designated doses of K116 every other day for 18 days. The tumor volumes and body weights of the mice were monitored every 2 days. The mice were sacrificed after treatment or if they met the humane endpoint criteria. The tumors were dissected, photographed, and weighed.22 The tumor tissues were collected and fixed in 10% neutral buffered formalin or stored at −80 °C for the following analyses.

2.12. Histological and immunohistochemical analyses

The xenograft tumor tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned using a semi-automated rotary microtome (RM2235; Leica). Their morphology was evaluated using hematoxylin and eosin (H&E) staining. The protein levels were analyzed by immunohistochemical (IHC) analysis using standard protocols with specific primary antibodies (STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), Ki-67). These sections were then deparaffinized and rehydrated. Incubation in 0.3% hydrogen peroxide was performed to block endogenous peroxidase activity. Following antigen retrieval with citrate buffer at a temperature of 95 °C, the sections were neutralized with endogenous peroxidases and obstructed with 2% BSA for a duration of 30 min. All assays were followed by staining with an HRP-conjugated secondary antibody. The stained slides were scored by two investigators according to the immunoreactive score of the Remmele and Stegner (IRS) system.23 The images were captured using an Olympus BX53 upright microscope.

2.13. Statistical analysis

Statistical analysis was performed using GraphPad Prism 10 software. The data were analyzed by one-way analysis of variance, followed by Tukey's post hoc test. The results were presented as the means ± SEMs and considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

3. Results

3.1. K116 displayed selective antiproliferative activity by inhibiting the phosphorylation of p-Y705STAT3 in breast cancer cell lines

The STAT3 CCD is essential for its SH2 domain-mediated receptor binding, and subsequent activation of STAT3 has emerged as a promising therapeutic target in breast cancer.16 Developing a potent and specific STAT3 CCD inhibitor could provide a strategic opportunity to suppress tumors associated with active STAT3.18 Previous studies identified the binding of K116 to the STAT3 CCD.19 We synthesized the K116 analogues, K134 and K114, to facilitate the pharmacological action and further investigation of the STAT3 CCD in breast cancer cells so as to avoid the chance of K116 inhibiting STAT3 activity (Fig. 1a and S1–S3†). The phosphorylated STAT3 plays indispensable roles in cancer cell growth and survival. We evaluated the anti-proliferative activity of K116, K134, and K114 against a panel of nine cancer cell lines originating from different tissue types to investigate the potential mechanisms underlying the efficacy of STAT3 allosteric inhibitors. Our results showed that the cell lines could be classified into two groups according to their sensitivity to K116, K134, and K114. As the results showed, MDA-MB-468 and 4T1cells were defined as sensitive to all three compounds, of which K116 at 10 μM inhibited the cell growth by more than 50%. The remaining cell lines exhibited moderate sensitivity to K116, K134, and K114 with inhibitory rates of less than 50%. Among all the tested compounds, K116 showed relatively strong selectivity and inhibitory activity on MDA-MB-468 and 4T1 cells (Fig. 1b–d and S4, Table S1–S3†).

Fig. 1. K116 selectively inhibited the proliferation of breast cancer cells depending on the inhibition of STAT3 activation. (a) Chemical structure of K116, K114, and K134. (b–d) K116, K114, and K134 selectively inhibited the proliferation of breast cancer cells. The cell proliferation was analyzed using the CCK8 assay. Half-maximal inhibitory concentrations were determined using GraphPad Prism10. (e) Western blot analysis with pY705STAT3 and STAT3 in 4T1, HGC-27, A549, and AC16 cells. All cells were treated with indicated concentrations of K116 for 24 h. (f) Inhibitory effect of K116 and Stattic on 4T1, HGC-27, A549, and AC16 cells. All cells were incubated with K116 or Stattic and the cell proliferation was measured using the CCK8 assay. The data are representative of three independent studies. The data are presented as the means ± SEMs, *P < 0.05, **P < 0.01.

Fig. 1

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4T1, HGC-27, A549, and AC16 cells were selected to detect the STAT3 activation so as to determine the dose–response relationship of K116. K116 reduced the proliferation of 4T1 in a dose-dependent manner by inhibiting the phosphorylation of p-Y705STAT3. However, it did not exhibit inhibitory activity in the proliferation of HGC-27 and A549 and had a minimal impact on p-Y705STAT3 phosphorylation in AC16 cells (Fig. 1e). K116 exhibited IC50 values of 4.8 μM and 15.2 μM for MDA-MB-468 and 4T1 proliferation, respectively, but showed no significant effect on the proliferation of other cells (Table S1†).

We compared the effects of K116 and Stattic (a STAT3 SH2 inhibitor) in inhibiting the proliferation of 4T1, HGC-27, A549, and AC16 cells to confirm its high selectivity. The result showed that K116 selectively inhibited 4T1 cells but not the others. Stattic inhibited the proliferation of all four cell types with IC50 ranging from 10.23 μM to 18.96 μM (Fig. 1f). These findings demonstrated that K116 selectively inhibited the proliferation of breast cancer cells by decreasing STAT3 activation and without altering p-Y705STAT3 phosphorylation leading to no effect on the proliferation of HGC-27, A549, and AC16 cells.

3.2. K116 directly and selectively targeted STAT3 in breast cancer cells

The CETSA experiment was performed to support the direct interaction between K116 and STAT3 so as to identify the potential target proteins of K116 in breast cancer cells (Fig. 2a). MDA-MB-468 cells were treated with K116 or DMSO and subjected to a CETSA heat pulse followed by soluble protein extraction and quantification. The results showed dose-dependent significant thermal stabilization of STAT3 upon treatment with K116 (Fig. 2b, c and S5†). K116 showed thermal stabilization on STAT3 and p-Y705STAT3 but not on Akt1, STAT1, and STAT5 (Fig. 2d and e and S6†). Collectively, these findings suggested that K116 might selectively and directly bind to STAT3 in breast cancer cells.

Fig. 2. K116 directly targeted STAT3 in breast cancer cells. (a) Scheme of the CETSA-WB experiment. (b) CETSA-WB experiment to further confirm the interaction between K116 (5 μM, 20 μM, and 25 μM) and STAT3. (c) The thermal stabilities of STAT3 with or without K116 (25 μM). (d) CETSA-WB experiment to further confirm the interaction between K116 (25 μM) and pY705STAT3, Akt, STAT1, and STAT5. (e) The thermal stabilities of pY705STAT3 with or without K116 (25 μM). (f) Docking model of K116 with the CCD. The crystal structure of STAT3 (PDB ID: 3CWG) was downloaded from the PDB. (g) Sequence alignment of the coiled-coil domain (CCD) of STAT3, STAT1, and STAT5.

Fig. 2

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Moreover, we performed docking analysis to further explore the selective binding of K116 with STAT3, and found that K116 formed a hydrogen bond with Gln202 and Asn175 of the STAT3 CCD based on our previous studies (Fig. 2f). STAT3, STAT1, and STAT5 belong to signal transducers and activators of the transcription family with high sequence homology in regions such as SH2 and SH3. Previous studies highlighted the crucial role of the residues STAT3 Asn175 and Gln202 within the CCD in recruiting STAT3 to both cytokine and growth factor receptors, leading to Tyr705 phosphorylation, dimer formation, nuclear translocation, and DNA binding. By aligning the amino acid sequences between the CCDs of STAT3, STAT1, and STAT5, Asn175 and Gln202 in STAT3 were identified as the nonhomologous sites, as Lys175 and Leu202 in STAT1, and Gln175 and Trp202 in STAT5 (Fig. 2g). The lack of conservation of the sequence between Gln202 and Asn175 is extremely critical for the action of K116. This finding explained why K116 affected the thermal stabilization of STAT3 compared to STAT1 and STAT5.

3.3. K116 inhibited STAT3 activation and attenuated the nuclear accumulation of pY705STAT3

To functionally validate the targeting of STAT3 by K116, the transcription activity and pY705STAT3 nuclear translocation were detected by luciferase activity analysis. The result also showed that K116 displayed a remarkable dose-dependent inhibitory effect on STAT3-induced luciferase activity (Fig. 3a). Moreover, we detected the activated pY705STAT3, pS727STAT3, STAT3, p-JAK, and JAK in the MDA-MB-468 cells to verify the predicted role of K116 in STAT3 activation. The results revealed that K116 decreased the phosphorylation of STAT3 at Ser-727 and Tyr-705, whereas the levels of total STAT3 protein, p-JAK, and JAK remained unchanged (Fig. 3b).

Fig. 3. K116 inhibited STAT3 transcriptional activity and pY705STAT3 nuclear accumulation. (a) K116 decreased the transcriptional activity of STAT3. STAT3 luciferase reporter activity was evaluated for treatment with increasing concentrations (6.25 μM, 12.5 μM, and 25 μM) of K116. (b) Western blots of pY705STAT3, pS727STAT3, and STAT3. (c and d) Immunofluorescence imaging of STAT3 nuclear localization in MDA-MB-468 cells treated with or without K116 (10 μM and 30 μM). pY705STAT3 (red) and DAPI (blue). The data represent three independent studies and are presented as the means ± SEMs, *P < 0.05, **P < 0.01.

Fig. 3

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We examined its effect on pY705STAT3 nuclear accumulation to investigate the potential role of K116. The results showed a significant reduction in the levels of pY705STAT3 at 10 μM and a notable decrease at 30 μM compared with the vehicle group in MDA-MB-468 cells (Fig. 3c and d). Altogether, these results indicated that K116 inhibited STAT3 activation functionally in cells.

3.4. K116 induced apoptosis and migration of breast cancer lines

We further determined whether K116 could decrease the viability of breast cancer cells by inducing apoptosis. The population of dead cells increased by more than threefold. Among the dead cells, apoptotic cell death increased by about fivefold after K116 treatment compared with that in the vehicle group (Fig. 4a and b). These findings indicated that K116 effectively drove the apoptosis of 4T1 cells.

Fig. 4. K116 induced apoptosis and migration of breast cancer lines. (a and b) K116 dose-dependently induced apoptosis, as detected by FACS analyses, followed by staining with PI and Annexin V in 4T1 cells treated with DMSO at concentrations of 1 μM and 25 μM. (c and d) MDA-MB-468 and 4T1 cells were treated with the vehicle (0.1% DMSO) or K116 (1 μM and 10 μM). Five fields were selected at random for photomicrographic images. The cells in each image were quantified. (e and f) Wound-healing test was performed after treatment with DMSO and 1 μM, 5 μM, and 10 μM K116 for 24 h, and the cells were visualized using a light microscope. The results represent three independent experiments. The data are presented as the means ± SEMs, *P < 0.05, **P < 0.01.

Fig. 4

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The migration and invasion of cancer cells into the bloodstream and surrounding tissues are crucial in cancer metastasis. Also, the transcription of target genes associated with these processes is regulated by STAT3 in the tumor microenvironment. We performed a Transwell assay on MDA-MB-468 and 4T1 cells to determine whether the decreased viability of breast cancer cells by K116 resulted from the migration and metastasis of breast cancer cells. K116 showed dose-dependent inhibition of invasion in MDA-MB-468 and 4T1 cells at 1 μM (Fig. 4c and d). In addition, K116 significantly suppressed the migration of 4T1 cells (Fig. 4e and f). Based on these results, we investigated the migratory properties of K116 in breast cancer cells.

3.5. K116 showed potent antitumor efficacy and inhibited tumor angiogenesis in breast cancer in vivo

The cell-based assays revealed a broad and robust antiproliferative effect of K116 on breast cancer cells. We generated a 4T1 murine breast cancer model to further evaluate the pharmacological effect of K116 in vivo. K116 treatment effectively suppressed tumor growth at 30 mg kg−1 without causing obvious weight loss (Fig. 5a–d), indicating the potent in vivo antitumor activity of K116. Moreover, H&E and IHC staining showed that breast cancer regression was associated with the reduced Ki-67 level, along with decreased levels of pY705STAT3 and pS727STAT3 (Fig. 5e and f and S8†). We concluded that K116 promoted the inhibition of the phosphorylation of STAT3 at Tyr-705 and Ser-727 and reduced the tumor angiogenesis ability. Overall, these results revealed that the inhibition of STAT3 by K116 attenuated the activation and angiogenic function of STAT3 in breast cancer.

Fig. 5. K116 displayed robust antitumor efficacy in 4T1 cell-derived xenografts. (a) Representative image of 4T1 xenograft tumors dissected from C57 mice treated intraperitoneally with the vehicle or 30 mg kg−1 K116 every other day for 18 days (n = 6). (b) Tumor weights in different groups of mice (n = 6). (c) Body weights in different groups of mice (n = 6). (d) Representative tissue sections from representative xenograft tumors stained with H&E. (e) Immunohistochemical analysis was performed for pY705STAT3, pS727STAT3, STAT3, and Ki-67 staining after treatment of xenograft tumors with the vehicle or K116. Representative images (×20 magnification) are shown. Scale bars = 200 μm. (f) Immunohistochemical analysis was performed for pY705STAT3, pS727STAT3, STAT3, and Ki-67. The data are presented as the means ± SEMs, *P < 0.05, **P < 0.01, and ***P < 0.001, ns, not significant.

Fig. 5

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3.6. Pharmacokinetic and safety study of K116 in vivo

Next, we detected the potential toxicity of K116. The effect of K116 on cell viability was investigated using CCK-8 in vitro. The results displayed low cytotoxicity and a negligible effect on the cell viability of AC16 and HEK-293 T cells (Fig. S4 and S9†). We also evaluated the biocompatibility and safety of K116 in vivo. The administration of K116 did not induce any significant pathological changes compared with that in the vehicle group, as revealed by H&E staining of major mouse organs (Fig. 6a). Moreover, the biochemical test of the liver and renal function conducted on blood samples demonstrated no significant differences compared with the vehicle group, indicating that K116 appeared to be biocompatible and passed the initial safety tests (Fig. 6b). During the treatment period, we found no significant loss in the weight of major organs and body weight in these days compared with those in the vehicle mice (Fig. 6c and d). In summary, these results demonstrated that the administration of K116 for a short duration of 15 consecutive days exhibited a significant level of safety in mice.

Fig. 6. Preliminary safety evaluation of K116. (a) H&E images of vital organs of mice after treatment with K116 (n = 5). H&E images of vital organs of mice 13 days after treatment with the vehicle (saline) and K116 (30 mg kg−1). Black scale bar = 50 μm. (b) Graph depicting the liver and renal function 13 days after the intraperitoneal injection of the vehicle and K116 (30 mg kg−1) (n = 5). (c) Weight of the vital organs of mice after treatment with the vehicle and K116 (30 mg kg−1) (n = 4). (d) Body weight of mice after treatment with the vehicle and K116 (30 mg kg−1) (n = 4). The data are presented as the means ± SEMs, *P < 0.05, **P < 0.01, and ***P < 0.001, ns, not significant.

Fig. 6

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The speculated network that inhibits breast cancer: ⊥ indicates an inhibitory effect

Above all, K116 exhibited effective binding to the STAT3 CCD and showed selective inhibition of STAT3 activation. These data suggest that the inhibition of STAT3 through binding to the allosteric domain CCD could be a promising therapy in breast cancer (Fig. 7).

Fig. 7. The speculated network of K116 on breast cancer. ⊥ indicates an inhibitory effect. The proposed mechanism of K116 inhibiting breast cancer through inhibition of the STAT3 signaling pathway.

Fig. 7

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4. Discussion

The JAK-STAT3 pathway plays a key role in the genetic and pharmacological control of cell biological processes. Further studies are required to determine the underlying reasons for the selective inhibition of pY705STAT3 by K116 in breast cancer cells rather than gastric cancer and lung cancer cells.24,25

In this study, we demonstrated that K116, our allosteric activator of the STAT3 CCD, showed favorable in vitro and in vivo anti-breast cancer activity by inhibiting pY705STAT3 and pS727STAT3, indicating that the pharmacological inhibition of the STAT3 CCD could be a promising therapy against breast cancer (Fig. 7). As previously mentioned, the phosphorylation of Ser727 was a functional posttranscriptional modification of STAT3.26 We assumed that K116 binding to the CCD affected the whole conformation of STAT3 and consequently interfered with the phosphorylation of STAT3 at Ser727 and Tyr705 residues. The inhibitors of the STAT3 SH2 domain demonstrated limited clinical activity because of the highly structurally homologous SH2 domain of STAT family members.27,28 Hence, targeting other domains of STAT3, including the CCD, has become of growing interest. As reported, the CCD of STAT3 is involved in the early activation of STAT3 and receptor binding. It serves as a crucial element in regulating early events during STAT3 activation and function, making it an indispensable component of the novel sequence. The CCD is an allosteric binding domain of STAT3 that regulates the conformation of SH2 so that STAT3 interacts with the receptor.17,18 Based on the structure, blocking the CCD can cause the structural rearrangement of STAT3 and hinder the dimerization of SH2, leading to the inactivation of STAT3. Although the relatively unique CCD plays a key role in activating STAT3, data regarding its function or potential as a drug target are limited. Therefore, targeting the CCD presents a novel therapeutic strategy for drug design and screening of small molecules that inhibit STAT3 activity by disrupting this domain to inhibit STAT3 activation for breast cancer.29–31

Further, we found that K116 could efficiently suppress the proliferation of MDA-MB-468 and 4T1 cells, having little effect on normal cells, such as HGC-27, A549, HEK-293 T, and AC16 cells. The STAT3 CCD was more sensitive to breast cancer therapy than the STAT3 SH2 domain due to the weak toxic effect of normal cells and sustained K116 treatment in vivo. STAT3 was regarded as a crucial regulator of angiogenesis under different pathological conditions by increasing EC survival, proliferation, and migration.32–34 The results showed that K116 reduced the Ki-67 level and decreased the levels of pY705STAT3, pS727STAT3, CD31, and VEGF and the number of vessels in tumor tissue in vivo, implying that the STAT3 pathway was further activated and amplified by the STAT3 inhibitor in resistant tumor angiogenesis (Fig. 5f and S7†). Thus, K116 showed potent antitumor efficacy and inhibited tumor angiogenesis in breast cancer in vivo. Therefore, we demonstrated that the binding of K116 with the STAT3 CCD was significantly against its proliferative effect, as well as its in vivo antitumor efficacy on breast cancer cells, thus showing the potent role of the STAT3 CCD in breast cancer. Our study suggested that the allosteric STAT3 CCD inhibitor K116 elucidated potent anticancer efficacy with high selectivity and low toxicity.

5. Conclusions

Overall, our findings demonstrated that K116, a STAT3 allosteric inhibitor, robustly selectively decreased STAT3-mediated proliferation, inhibited migration and gene transcription, and induced apoptosis in breast cancer cells. K116 exhibited effective binding to the STAT3 CCD and showed selective inhibition of STAT3 activation in vitro and in vivo. These results provided pharmacological evidence to support the exploration of the antitumor efficacy by inhibiting the STAT3 CCD, thereby endorsing a preclinical strategy with promising potential for developing novel STAT3 inhibitors to defeat breast cancer. By combining pharmacological approaches, we demonstrated that the inhibition of STAT3 through binding to the allosteric domain CCD could be a promising therapy in breast cancer.

Ethics statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Northwestern Polytechnical University and approved by the Animal Ethics Committee of the Lab of Animal Experimental Ethical Inspection of Northwestern Polytechnical University (Permit No. 202301130).

Author contributions

Min Huang contributed to the conceptualization, formal analysis, methodology, software, visualization, and writing of the original draft. Wei Wang was involved in the conceptualization, data curation, formal analysis, methodology, software, and writing of the original draft. Liyue Cao was involved in the conceptualization, data curation, formal analysis, methodology, software, and writing of the original draft. Jiaxin Liu carried out the data curation, methodology and software. Can Du carried out the methodology and software. Jian Zhang and Min Huang were involved in the conceptualization, data curation, funding acquisition, supervision, and writing, review and editing of the manuscript. All authors have approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-016-D4MD00926F-s001

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81803031), the Natural Science Foundation of Shaanxi Province (No. 2024JC-YBQN-0863) and the Guiding Program of Health System of Qinghai Province (No. 2024-wjzdx-08). We thank Professor Zhijun Zuo for synthesizing compound K116.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4md00926f

Data availability

The data supporting this article have been included as part of the ESI.†

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-016-D4MD00926F-s001
MD-016-D4MD00926F-s001.pdf (1.4MB, pdf)

Data Availability Statement

The data supporting this article have been included as part of the ESI.†

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