STAT3: Key targets of growth-promoting receptor positive breast cancer

Abstract

Breast cancer has become the malignant tumor with the first incidence and the second mortality among female cancers. Most female breast cancers belong to luminal-type breast cancer and HER2-positive breast cancer. These breast cancer cells all have different driving genes, which constantly promote the proliferation and metastasis of breast cancer cells. Signal transducer and activator of transcription 3 (STAT3) is an important breast cancer-related gene, which can promote the progress of breast cancer. It has been proved in clinical and basic research that over-expressed and constitutively activated STAT3 is involved in the progress, proliferation, metastasis and chemotherapy resistance of breast cancer. STAT3 is an important key target in luminal-type breast cancer and HER2-positive cancer, which has an important impact on the curative effect of related treatments. In breast cancer, the activation of STAT3 will change the spatial position of STAT3 protein and cause different phenotypic changes of breast cancer cells. In the current basic research and clinical research, small molecule inhibitors activated by targeting STAT3 can effectively treat breast cancer, and enhance the efficacy level of related treatment methods for luminal-type and HER2-positive breast cancers.

Introduction

Breast cancer (BRCA) incidence and mortality rates are rapidly rising worldwide, making it the most common malignant tumor in women since 2021, accounting for over 30% of all newly diagnosed cancers [1]. According to the American Cancer Society, approximately 281,500 new breast cancer cases were recorded in the United States in 2021, with an estimated 43,600 deaths. According to the prediction model, it was estimated that 287,850 new cases of breast cancer and approximately 4,3250 deaths occurred in the United States in 2022 [2]. In most developing and developed countries, cancer is among the leading causes of "premature death" among residents [3]. The term "premature death" refers to the death of adults aged between 30 and 70. Breast cancer is classified into three molecular types based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor (HER2) in breast cancer cells: ER- or PR-positive (also known as luminal breast cancer), HER2-positive breast cancer, and triple-negative breast cancer (TNBC) (ER-/PR-/HER2-) [4]. Luminal type B breast cancer can also be categorized as HER2-positive or HER2-negative based on the expression of HER2. HER2-positive and luminal breast cancers are also known as growth-promoting receptor-positive breast cancers because of the presence of certain genes (ER, PR and/or HER2) that drive tumor cell proliferation. growth-promoting receptor-positive breast cancer accounts for approximately 75% of all breast cancer cases [5]. Although tumors in patients with triple-negative breast cancer (TNBC) are characterized by poorer histological grading and shorter survival times, the tumors with growth-promoting receptors still exhibit higher rates of distant metastasis and higher tumor proliferation indices compared to TNBC [6].While drugs targeting growth-promoting receptors (such as anti-HER2 drugs and estrogen receptor antagonists) have improved the prognosis for patients with growth-promoting receptor-positive breast cancer, the issue of treatment resistance continues to significantly affect the overall therapeutic outcomes in this group of patients [7]. Although conventional therapies can achieve significant tumor elimination in patients with breast cancer harboring positive growth-promoting, a variety of treatment-related adverse reactions (surgical trauma, high cost, drug resistance, and cytotoxicity in normal tissues) are closely related to the risk of treatment interruption [8]. These adverse reactions may lead to discontinuation of treatment or insufficient doses, resulting in tumor recurrence and metastasis [9]. Therefore, new therapeutic targets and methods are essential for improving the survival rate, quality of life, and treatment tolerance of breast cancer patients.

STAT3 is an important protein for the growth, survival, differentiation, regeneration, immune response, respiration, metabolism, and other fundamental cellular functions of breast cancer [10]. STAT3 expression and subcellular localization are regulated by upstream signaling molecular proteins, such as Janus kinase (JAK) and epidermal growth factor receptor (EGFR) [11]. The activation of these proteins results in STAT3 localization to the nucleus or mitochondria. STAT3 in the nucleus combines with target DNA to promote DNA transcription to become the corresponding protein, while STAT3 entering mitochondria promotes morphological or functional changes. The entry of STAT3 into the mitochondria, resulting in alterations in mitochondrial protein expression and structural changes (including increased mitochondrial membrane permeability and damage to mitochondrial cristae), is one of the key factors triggering inflammation associated with tumor cells and metabolic reprogramming in these cells. STAT3 plays a stable regulatory function in maintaining the stability of normal tissue cells [12]. In tumor cells, STAT3 is activated and/or mutated by different components of the tumor microenvironment where tumor tissue is located, resulting in uncontrolled proliferation, invasion, and metastasis of tumor tissue [13]. High STAT3 expression in all types of breast cancer patients is closely related to treatment resistance and reduced survival [14]. STAT3 activation is a key factor in the formation, proliferation, metastasis, recurrence, and drug resistance of breast cancer and is an important marker of its poor prognosis [15]. Therefore, the STAT3 pathway is a promising therapeutic target for breast cancer. Current research methods for regulating STAT3 activation with STAT3 inhibitors have mainly focused on the critical role of STAT3 in regulating breast cancer cell proliferation, apoptosis, angiogenesis, treatment resistance, and metastasis [16]. Therefore, the STAT3 signaling pathway is a promising therapeutic target for growth-promoting receptor-positive breast cancer.

The structure and biological function STAT3

STAT3 consists of six domains [17, 18]: (1) N-terminal domain (NTD), which stabilizes STAT3 entry into the nucleus and DNA binding; (2) Spiral coil structure domain (CCD), which recognizes specific DNA sequences to form STAT3-DNA complexes; (3) DNA binding domain (DBD), which recognizes specific DNA sequences to form STAT3-DNA complex; (4) linker domain (LD), which is involved in the transcriptional activation process of cells. (5) Src homologous 2 domain (SH2), which recognizes and binds to the docking site of phosphotyrosine residues on the kinase receptor, places STAT3 near the active JAK, resulting in the phosphorylation of STAT3 by tyrosine kinases, in which SH2 is also critical for the dimerization of two STAT3 monomers; (6) C-terminal transactivation domain (TAD), which contains a serine residue (Ser727) necessary to regulate the maximum transcriptional activity of genes. Additionally, STAT3 activation depends on two phosphorylation sites: the tyrosine residue on SH2 (Tyr705) and the serine residue on TAD (Ser727). Different phosphorylation sites lead to diverse subcellular localization and transcription levels of STAT [19]. The tyrosine residue (Tyr705) in SH2 mainly promotes nuclear translocation of STAT3 [20]. Recent studies have found that tyrosine residues (Tyr705) and serine residues on TAD (Ser727) also cause STAT3 to move to mitochondria. Each domain of STAT3 plays a different role in signal transduction and gene transcription activation [21]. STAT3 contains a traditional nuclear localization signal (NLS) that facilitates its transport into the nucleus [22]. The nuclear access of STAT3 is indeed mediated by members of the importin family, which are responsible for recognizing the NLS and transporting STAT3 through the nuclear pore complex [23]. Once in the nucleus, STAT3 can regulate gene expression by binding to specific DNA sequences and interacting with other transcription factors. Therefore, drugs based on the STAT3 domain hallmark have been used to improve the affinity of selective inhibitors for targets (Fig. 1).

Fig. 1

Schematic diagram of STAT3 protein structure. Schematics of STAT3 protein structure and function. A The STAT3 protein structure includes an N-segment domain, helix-helix domain, DNA binding domain, linker, Src homology 2 domain, and C-segment domain. The DNA-binding domain of the STAT3 protein is primarily responsible for DNA transcription, while the Src homology domain is primarily responsible for promoting STAT3 protein dimerization. These domains are regulated by the phosphorylation level of Y705 and S727 sites. B The three-dimensional structure diagram of STAT3 protein promotes it's role in DNA binding, dimerization, and reverse transcription

STAT3 has two main isoforms: full-length STAT3α (770 amino acids, 92 kDa) and truncated STAT3β (722 amino acids, 83 kDa); both are generated by the alternative splicing of exon 23, with STAT3α being the main splicing form. Compared with the full-length STAT3α, STAT3β lacks the TAD domain and thus does not possess the Ser727 phosphorylation site [24]. However, it has a tail of seven amino acid residues. The two isoforms have distinct STAT3 domains; therefore, their biological behaviors are also different [25]. Constitutive activation of STAT3α plays a vital role in carcinogenic pathways and is an essential oncogenic factor [26]. The expression levels of STAT3α and STAT3β show minimal differences in normal tissue cell types [27]. However, in tumor cells, the expression levels of STAT3α and STAT3β exhibit a certain degree of variation, with STAT3α being expressed at significantly higher levels than STAT3β. This difference is likely closely related to STAT3α's primary involvement in gene transcription [28]. STAT3β inhibits the constitutive activation of STAT3α and participates in the transcription of specific cancer suppressor words, such as inhibiting the growth of melanoma, breast, and lung cancer and promoting their apoptosis [29]. However, STAT3α expression level is significantly higher in all tumor tissues than STAT3α; thus, STAT3 can be considered an evident cancer-related protein. In normal cells, STAT3α primarily promotes cell proliferation, survival, and differentiation, and is involved in cellular signal transduction and immune responses. In contrast, STAT3β exerts inhibitory effects, suppressing the activity of STAT3α and promoting the expression of certain tumor suppressor genes. This functional dichotomy highlights the important role of STAT3β in regulating cell growth and maintaining cellular homeostasis. The balance between these two isoforms is crucial for the proper functioning and health of normal cells [30]. STAT3 activation can be caused by the interaction of polypeptide ligands of related cell growth factors. These growth factors contribute to cell growth, reproduction, migration, and apoptosis by activating STAT3 [31]. These growth factor ligands bind to their corresponding receptors on the cell surface to attract Src and JAKs. Simultaneously, activation of the tyrosine residue in the corresponding receptor activates STAT3. Inflammatory factor receptors involved in the inflammatory response, such as IL-3 and IL-6 receptors, generally have gp130 subunits [32]. When cytokines bind to gp130, they trigger the signal activation of another receptor subunit, gp130. This process subsequently recruits downstream JAK1 and JAK2, initiating their phosphorylation. The activated JAK2, in turn, phosphorylates tyrosine residues on the cytoplasmic domain of the IL-6 receptor. These residues serve as docking sites for the SH2 domain of STAT3, leading to the phosphorylation (at Tyr705) and activation of STAT3 homodimers. Activated Src and JAKs can promote the SH2 and TAD domains of STAT3 and the activated complex residues and serine residues on the corresponding receptors to combine with each other [33]. Simultaneously, this binding process requires the phosphorylation of specific influencing factor receptors by tyrosine kinases or proteins with tyrosine kinase activity [34]. The activation region of the partially activated STAT3 protein consists of serine residues; therefore, some STAT3 proteins that initially existed in the cytoplasm are stimulated and activated, detach from their receptors, form dimers through their SH2 domains, enter the nucleus or mitochondria, and combine with the corresponding DNA fragments [22]. This process of self-dimerization is also responsible for drug resistance in some targeted therapeutic drugs [35]. Consequently, STAT3 regulates the activity of breast cancer cells by transforming short-term activation signals into changes in gene expression.

Signal pathway and regulation of STAT3

The activation of canonical STAT3 is initiated by the phosphorylation of cytokine receptors, including IL-6 receptor, tyrosine kinase-related receptors such as JAK, tyrosine kinase (RTK), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), represented by EGFR, and non-receptor tyrosine kinases such as Src and Abl [36]. Moreover, microRNA, toll-like receptors, and G protein-coupled receptors (GPCR) can activate STAT3 [37].

Upregulation of upstream signaling pathways is attributed to molecules produced in the tumor microenvironment; it causes constitutive activation of STAT3 and promotes cell proliferation (such as cyclin D-1, c-Myc), apoptosis (such as Bcl-2, survivin), immunosuppression (such as IL-10), inflammation (such as COX-2, IL-6, IL-17A), invasion and metastasis (such as vimentin, matrix metalloproteinases), angiogenesis (such as vascular endothelial growth factor (VEGF), HIF-1α, hepatocyte growth factor (HGF)), and other downstream target genes to promote the occurrence and development of cancer jointly [38]. Among them, cytokines, growth factors, and angiogenic factors (such as IL-6, IL-10, VEGF) encoded by many downstream target genes of STAT3 reactivate STAT3 signaling, forming a feed-forward autocrine feedback loop that provides conditions for the constitutive activation of STAT3 in tumors [39]. This feedback loop is essential for controlling viral infection and mediating antitumor immunity.

Cytokine receptors usually lack intrinsic tyrosine kinase activity; thus, they rely on tyrosine kinase-related receptors (JAK is the most classic). IL-6 is a traditional activator of STAT3 and a vital driver of JAK-STAT3 signaling pathway activation in many tumors [40]. After binding to the ligands, IL-6 family receptors induce the homodimerization or heterodimerization of gp130 [41]. The gp130 receptor complex dimerizes to recruit and bind JAK, which is phosphorylated on tyrosine residues at the end of the cytoplasmic domain of the receptor complex, providing a site for the SH2 domain of STAT3, leading to the recruitment of STAT3 [42]. Subsequently, STAT3 bound to the site is activated by neighboring JAK by phosphorylating specific tyrosine residues at it's C terminus. In the cell, STAT3 typically exists in an inactive state in the cytoplasm. Upon activation by cytokines, tumor growth factors, and other signaling molecules that bind to their respective receptors, JAK (Janus kinase) phosphorylates the tyrosine residue (Y705) of STAT3, converting it into an active form. Phosphorylated STAT3 dimerizes, and the active STAT3 dimers possess a nuclear localization signal (NLS) that facilitates their transport to the nucleus by interacting with receptors in the nuclear membrane. Once inside the nucleus, STAT3 binds to specific DNA sequences to regulate the transcription of target genes, thereby influencing processes such as cell growth, differentiation, and apoptosis. Receptor tyrosine kinases Src and Abl can also activate STAT3, and phosphorylated STAT3 monomers form homodimers with other phosphorylated STAT3 monomers, or phosphorylated STAT1 forms heterodimers, which is transferred to the nucleus and then recognize (TTCN3GAA) on the promoter region of the target gene, producing cascade gene transcription [43]. Additionally, RTKs catalyze STAT3 phosphorylation via intrinsic tyrosine kinase activity of STAT3 in the receptor [44]. In addition to STAT3 phosphorylation at Y705 and S727, there are other post-translational modifications, such as methylation, ubiquitination, acetylation, S-glutathionylation, and S-nitrosylation, which are linked to STAT3 transcriptional activity, dimerization, translocation, and degradation [45]. The oncogenic properties of STAT3 are closely linked to its nuclear pathway. Upon entering the nucleus, STAT3 can activate genes associated with cell proliferation, survival, and apoptosis, such as Bcl-2 and Cyclin D1, thereby promoting tumor cell growth and resistance to apoptosis. Within the nucleus, STAT3 regulates the expression of pro-inflammatory factors, shapes the tumor microenvironment, inhibits anti-tumor immune responses, and facilitates tumor growth and metastasis. In many cancers, STAT3 exhibits persistent phosphorylation and activation, and this aberrant state allows for the continuous regulation of downstream target genes, exacerbating tumor progression.

Under physiological conditions, the activation of STAT3 signaling pathway is short-lived and can be quickly restored to it's basic state to prevent the regulation of unplanned genes that cause many diseases in humans, mainly including four negative regulatory factors: inhibitors of cytokine signaling (SOCS), activated STAT protein inhibitor (PIAS), protein tyrosine phosphatase (PTP), and ubiquitin–proteasome pathway [46]. For example, SOCS3 is an important downstream target gene of JAK/STAT3, and it inhibits JAK1, JAK2, and TYK2, which are important for the proteasome degradation of the gp130 receptor complex, thereby participating in the negative feedback regulation of the STAT3 signal in the normal steady state [47]. Inhibiting or decreasing these negative regulatory factors can lead to constitutive activation of STAT3, usually observed in tumor patients (Fig. 2).

Fig. 2

Main STAT3 signal transduction pathways in breast cancer. STAT3 is an essential regulatory protein in breast cancer. A IL-6 (EGFR)/STAT3 signaling regulates breast cancer proliferation, invasion, anti-apoptosis, and other malignant biological behaviors. The occurrence of these phenotypes depends on the overactivation of STAT3. B The effects of different upstream receptors on the activation of the STAT3 signaling pathway and the regulation of STAT3 by other proteins in different cellular localizations

STAT3 is an important oncogene in breast cancer

Cancer-related genes play a decisive role in the occurrence and development of breast cancer. For example, K-ras and Src are typically activated breast cancer-related genes that play a very important function in the occurrence and development of breast cancer [48]. STAT3 has been identified as an important oncogene in growth-promoting receptor-positive breast cancer.

More than 70% of cancer patients have overexpression, abnormal phosphorylation, and structural changes of STAT3, with non-small-cell lung cancer and breast cancer being the most common [49]. STAT3 expression levels in breast cancer tissues were significantly higher than in normal and benign breast tissues [50]. STAT3 expression activation is usually transient in normal tissues. STAT3 is persistently highly expressed in breast cancer patients. STAT3 expression was significantly higher in luminal and HER2-positive breast cancers than in TNBC [51]. However, in some studies, the expression level of STAT3 in triple-negative breast cancer is the highest among all molecular subtypes of breast cancer. This may be closely related to the patient's treatment history [52, 53]. However, the expression level of STAT3 is closely related to the treatment benefits in patients with luminal-type breast cancer and HER2-positive breast cancer. Therefore, STAT3 can be considered a key oncogene in growth-promoting receptor-positive breast cancer. The higher expression levels of STAT3 indicate more STAT3 activation, which also implies increased transcription of proteins related to breast cancer cell proliferation and invasion. This directly leads to a poorer prognosis in breast cancer patients. Interestingly, sufficiently high levels of STAT3 expression can bypass the need for upstream protein-induced phosphorylation. High expression of STAT3 can promote its autophosphorylation and dimerization, and this self-dimerized form can likewise translocate into the nucleus to transcribe proteins associated with the proliferation and invasion of breast cancer cells, facilitating tumor growth and spread. The activation pathways of STAT3 in solid tumors involve multiple cell signaling pathways, including the classical JAK/STAT pathway, Src and other non-receptor tyrosine kinases, GPCR, RAS/MAPK, PI3K/AKT, oxidative stress, and others. The interplay among these pathways collectively regulates STAT3 activity, thereby influencing tumor cell proliferation, anti-apoptosis, invasion, and immune evasion properties.

STAT3 is overexpressed in tumor tissues and self-activated to form dimers that promote cell carcinogenesis and cause tumors in animals. The C-terminal loop (STAT3-C) is replaced by two cysteine residues in the SH2 domain of STAT3, resulting in self-dimerization that can occur without tyrosine phosphorylation. After transfection of immortalized cell lines with STAT3-C, cells undergo spontaneous clonogenesis. Tumor tissues appeared in nude mice following STAT3-C intervention [22].

Abnormal STAT3 phosphorylation levels are also significant in tumorigenesis, development, and metastasis. The analysis of clinical samples revealed that high STAT3 expression is closely related to drug resistance to targeted therapy, endocrine therapy, and chemotherapy in breast cancer patients [54]. After studying tumor tissues from patients with breast cancer obtained during surgery, STAT3 expression was found to be significantly associated with the overall survival of patients. Patients with higher STAT3 expression levels and a positive proportion of breast cancer tissues have shorter progression-free survival and overall survival and are less sensitive to various antitumor treatments [55]. In some studies, STAT3 expression levels were identified as an important indicator of the prognosis of breast cancer patients [56]. In some hematological tumors, high STAT3 expression leads to increased programed death ligand-1 (PD-L1) expression, prompting hematological tumor cells to evade immune cell attack. As a result, higher STAT3 expression levels in hematological tumors can also lead to shorter overall survival times and reduced treatment responses [57].

STAT3 inhibitors, in basic experiments, have been proven to inhibit tumor cell proliferation while effectively promoting apoptosis. Using STAT3 inhibitors, Bcl-xL expression can be downregulated to inhibit tumor cell proliferation and promote apoptosis. STAT3 can also promote tumor cell metastasis in patients with tumors by regulating downstream proteins (MMP-2 and MMP-9) [10]. Therefore, STAT3 also plays a crucial role in cancer recurrence and metastasis. Tumor cell proliferation and metastatic activities were significantly reduced following the knockdown of STAT3 upstream molecules or STAT3 inhibitor treatment. STAT3 promotes luminal breast cancer progression by regulating the expression of estrogen-related proteins. Thus, STAT3 is a key link in resistance to endocrine therapy and adverse reactions in luminal breast cancer, promoting the occurrence of poor prognosis events [58]. Targeting STAT3 can block the proliferation and metastasis of ER-positive breast cancer and improve the sensitivity to endocrine therapy. As a result, various components of the breast cancer-related tumor microenvironment can promote and maintain high STAT3 expression and it's sustained activation in breast cancer cells, leading to breast cancer formation, progression, and poor prognosis [59].

However, some studies disagree on the significance of STAT3 in promoting tumor cell proliferation, invasion, and metastasis in breast cancer. In a study of oral squamous cell carcinoma, the expression levels of IL-6 and STAT3 were inconsistent. This result contradicts the generally accepted view that STAT3 is primarily activated by the IL-6/JAK pathway. These data also confirmed that the high expression and sustained activation of STAT3 in tumor cells is mediated more by the EGFR/Src pathway than the IL-6/JAK pathway [60]. IL-6 promotes vascular invasion, reduces the 5-year disease-free survival rate, and induces the activation of naive T cells into cytotoxic T cells. Therefore, IL-6 must be expressed at a certain level in the tumor microenvironment to maintain the activation and attack of T cells [61]. The 5-year overall survival rate of head and neck cancer patients with high STAT3 expression was 72.4%. In patients with low STAT3 expression, the 5-year overall survival rate was 38.3%. This may be closely related to STAT3 expression and the efficacy of some targeted therapies [62]. Basic experiments revealed that STAT3 overexpression inhibited tumor cell apoptosis and promoted the formation and development of tumor cells. Protein chip analysis of lymph node-negative and -positive breast cancer found that patients with higher phosphorylated STAT3 (Tyr705) expression had longer short-term and long-term survival times [63]. This result also demonstrated that patients with STAT3-positive breast cancer have a better prognosis. A study confirmed that the ARF-MDM2-p53 tumor suppressor axis is regulated by IL-6/STAT3. However, the risk of prostate cancer metastasis and recurrence was significantly increased in the mouse model following IL-6/STAT3 inhibitor treatment. This also demonstrated that IL-6/STAT3 signaling promoted the recurrence and metastasis of tumors and inhibited the proliferation of tumor cells to a certain extent [64].

Despite the conflicting studies on the role of STAT3 in other types of cancer, this does not prevent STAT3 from acting as a significant oncogene in breast cancer. In growth-promoting receptor-positive breast cancer, high levels of STAT3 expression still represent poorer treatment outcomes and shorter survival times. Although a small number of studies have shown opposite treatment results, this may be due to insufficient sample sizes.

Overall, STAT3 is an important oncogene in breast cancer, particularly in growth-promoting receptor-positive breast cancer. Different STAT3 expression levels have varying effects on therapeutic drug sensitivity and survival prognosis in breast cancer patients. Although high STAT3 expression results in shorter overall survival time for patients, it also indicates a higher treatment response rate. In HER2-positive breast cancer patients, HER2 phosphorylation can activate different signaling pathways, and STAT3 can directly enter the nucleus to participate in cell physiological functions. Accordingly, a higher STAT3 level indicates that the signaling mode after HER2 phosphorylation is relatively single, implying that anti-HER2 treatment produces a better therapeutic effect [65] (Fig. 3).

Fig. 3

Regulation of STAT3 in HER2-positive breast cancer. In HER2-positive breast cancer, STAT3 is activated through the EGFR/Src and IL-6/JAK pathways. This leads to abnormally high-level expression of STAT3 in HER2-positive breast cancer. Currently, HER2 inhibitors (trastuzumab, pertuzumab, and lapatinib) are primarily used to treat HER2-positive breast cancer. These drugs can block STAT3 activated by the EGFR/Src pathway but cannot inhibit STAT3 activated by IL-6/JAK pathway. The sustained high level of STAT3 expression also enhances the resistance of HER2-positive breast cancer cells to HER2 treatment

STAT3 activation leads to sustained activation of downstream signaling molecules and other signaling pathways

STAT3, an important transcription factor in breast cancer, regulates the target gene expression levels of several components of traditional Chinese medicine in the tumor microenvironment of breast cancer. The activation of STAT3 signaling can up regulate the expression of essential breast cancer-related growth-promoting factors while downregulating or silencing the expression of breast cancer-related tumor suppressor genes [66]. The regulation of STAT3 on breast cancer-related target genes promotes signaling related to breast cancer occurrence and development. Specific downstream targets of STAT3 can clarify the phenotypic changes in breast cancer cells following STAT3 mutation and/or overexpression. When STAT3 is activated to promote signaling, breast cancer cells acquire the basic abilities of tumor cells, such as proliferation, invasion, metastasis, immune escape, apoptosis resistance, and metabolic reprogrammingt [67]. In normal tissue cells, STAT3 activation is always transient. STAT3 activation is strictly regulated by the SOCS3. SOCS3 binds to the STAT3 promoter, blocking STAT3 from entering the nucleus and binding to the DNA sequence of target genes through negative feedback regulation. SOCS3 expression levels are significantly lower in breast cancer cells than in normal breast epithelial cells, and STAT3 cannot be negatively regulated to promote STAT3 inactivation after activation during breast cancer development [50]. Consequently, STAT3 signaling in breast cancer cells remains a sustained high-level activation. The overexpression of STAT3 can also promote the expression of related oncogenes in breast cancer cells through it's positive feedback regulation to continuously maintain the high level of STAT3 expression. For example, the expression level of NF-κB increased after STAT3 overexpression in TNBC cell lines. STAT3 can convert NF-κB recruited to the promoter of the fascin gene FSCN1 to form an active transcriptional complex. The expression of FSCN1 can promote the remodeling of breast cancer cell morphology, reduce adhesion, and increase the movement of breast cancer cells, leading to the metastasis of breast cancer cells [68].

The phosphorylation of Tyr705 and Ser727 is necessary to activate STAT3 and promote it's regulation of downstream target genes. STAT3 is the central borrowing point of signaling pathways involved in inflammation, immunity, and proliferation, including IL-6/IL-11, EGFR, and ERK signaling. The interaction of these signaling pathways highlights the important role of STAT3 in promoting tumor development. After phosphorylation, dimerization, and translocation to the nucleus, STAT3 binds to consensus-binding sequences in the promoter region of target genes to regulate their transcriptional activity. The primary binding site of STAT3 in breast cancer cells is a gene promoter that regulates cell motility, growth, proliferation, and inflammation. STAT3 can play a key role in regulating the invasion and metastasis of breast cancer cells by transcriptionally regulating the proteins of pathways related to extracellular matrix organization, extracellular structure organization, collagen metabolic process, anchor junctions, adherens junctions, and movement regulation [69].

Phosphorylated STAT3 can interact with GLI1, a glioma-associated oncogene. Such interactions can be observed in patients with TNBC and HER2-positive breast cancer. The co-activator protein of STAT3/GLI1 was found in many lymph node metastases. Patients with higher STAT3/GLI1 complex levels have worse long-term survival outcomes. The influence of this protein complex on prognosis can also be confirmed in non-small cell lung cancer and chronic lymphocytic leukemia. The analysis of genes co-activated by STAT3 and GLI1 using CHIP-seq revealed that the co-overexpression of STAT3/GLI1, R-Ras2, Cep70, and UPF3A was significantly upregulated. These genes have been confirmed to be closely related to PI3K signaling and microtubule disorders. High expression of these genes reduces the disease-free survival rate of patients with TNBC and HER2-positive breast cancer [70].

The interaction of STAT3 with other transcription factors and the regulation of other signaling pathways are important research directions for the clinical analysis of STAT3. Studies have found that STAT3 regulates many genes related to immune escape and chemoresistance, such as TGF-β, VEGF, NF-κB, OCT-4, and c-MYC. In a preclinical study, combining a PD-1 antibody with a STAT3 inhibitor enhanced antitumor responses in a colon cancer model [71]. This result demonstrates that STAT3 inhibitors enhance the antitumor effects of PD-1 antibodies. Although identifying a new antitumor mechanism using the combination of STAT3 inhibitors and PD-1 antibodies is impossible, further research is needed to evaluate the efficacy and safety of STAT3 inhibition combined with immunotherapy in patients with tumors [72]. However, the comprehensive crosstalk between STAT3-dependent pathways and immune checkpoints represents an attractive therapeutic target for breast cancer. This will likely provide a new treatment and research idea for encouraging gene-positive malignant tumors to use immunotherapeutic drugs for antitumor treatment.

STAT3 promotes the development of growth-promoting receptor-positive breast cancer from multiple perspectives

All cancer cells have unique physiological characteristics. Malignant tumors have some hallmarks that distinguish them from normal tissue cells. It has been confirmed that these features cause tumor tissue to proliferate continuously, invade, and metastasize, eventually leading to death. Hanahan et al. (2000) proposed a sign that tumor cells are different from normal cells, stating that they contain six necessary properties for inducing malignant cell growth, including sustaining proliferative signaling, evolving growth suppressors, resisting cell death, enabling replicative mortality, inducing or accessing vascular, and activating invasion and metabolism [73]. With the advancement of cancer biology research, the number of basic markers of tumor cells increased to 10 by 2011. Four new markers were added: genome stability and mutation, tumor-promoting inflammation, cellular metabolism-regulating, and avoiding immune destruction [74]. In 2022, four new cancer markers were added to the tumor cell marker list: unlocking phenotypic plasticity, non-mutational epigenetic reprogramming epigenetic reprogramming, senescent cells, and polymeric microorganisms. The phenotypes of these tumor-related features are caused by related signal pathway changes [75]. STAT3, a tumor-causing signaling pathway, is an important tumor-promoting factor. Studies have also confirmed that STAT3 plays a crucial role in the formation, development, and metastasis of growth-promoting receptor positive breast cancer [76]. Additionally, inhibiting STAT3 can be a promising therapeutic strategy for growth-promoting receptor-positive breast cancer (Table 1, Fig. 4).

Table 1 Biological role of STAT3 in HER2 positive breast cancer and luminal breast cancer

Fig. 4

Regulation of STAT3 on basic hallmarks of tumors. Schematic of the regulatory effect of STAT3 on cancer markers. Cancer has 14 basic characteristics, and STAT3 has an essential influence on 13 of these (maintaining proliferation signal, evolving growth inhibitory factor, deregulating cell metabolism, avoiding immune damage, resisting cell death, achieving immortal reproduction, genome instability and mutation, tumor-promoting inflammation, inducing or approaching blood vessels, activating invasion and metastasis, unlocking phenotypic plasticity, non-mutant epigenetic reprogramming, and aging cells)

STAT3 is an important regulatory factor for cell cycle regulators

Tumor cells are distinguished by their unrestricted proliferation, closely linked to their uncontrolled cell cycle. The deregulation of cancer-related genes and proteins, such as STAT3, leads to abnormal cell cycle. Abnormal expression levels and/or mutations in cell cycle-related proteins lead to sustained cyclin expression in tumor cells, thereby promoting cell replication and proliferation [77]. In growth-promoting receptor-positive breast cancer cells, the high expression of some surface proteins (HER2 and ER) that promote tumor cell proliferation activates the initiation of cyclins in the nucleus, prompting breast cancer cells to remain in the replication cycle for a long time [78].

HER2 is an important protein in the EGFR family. Although HER2 does not have a specific ligand, it can form homodimers with itself (HER2-HER2), which is particularly common in HER2-overexpressing cells [79]. HER2 can also form heterodimers with other members of the EGFR family (such as HER1, HER3, and HER4). The formation of these heterodimers is often caused by the activation of ligands (EGF, fibroblast growth factor, (FGF), and so on) of other members of the family [80]. Activated HER2 can initiate multiple signaling pathways that play key roles in cell growth, differentiation, and survival, such as Ras/Raf/MAPK, PI3K/Akt, JAK/STAT, PLCγ pathways, and other downstream signaling pathways of HER2. The activation of these signaling pathways promotes the expression of cell cycle-related proteins in cells. The ER is an estrogen-dependent nuclear transcription factor [81]. ERs are generally located in the nucleus but can also be expressed in the cell membrane and cytoplasm. After activation, the ER activates downstream MAPK, PI3K, and other signaling pathways to promote cell proliferation. The overexpression of HER2 and ER promotes the continuous activation of cell proliferation-related signaling pathways and promotes the continuous proliferation of cells [82]. Consequently, STAT3 is continuously activated in the nucleus of HER2-positive and luminal breast cancer cells to participate in cell replication and protein transcription.

Many intracellular proteins in HER2-positive breast cancer affect the expression and physiological function of STAT3, and HER2 overexpression affects STAT3 through different signaling pathways to promote breast cancer cell proliferation. Overexpression of HER2 activates the Ras/MAPK and JAK/STAT3 signaling pathways to promote breast cancer cell proliferation via Ki-67 [83]. ANO1/CLC-3 regulates intracellular Cl⁻ to participate in HER2 transcription and mediates the PI3K/Akt/mTOR and/or STAT3 signaling pathways in HER2-positive breast cancer cells [84]. A proteomic assay of HER2-positive breast cancer cells revealed high phosphorylation/activation levels of NK and STAT5A/B, ERK/MAPK, STAT3, CREB, p70 S6 kinase, IKBA, and p38 [85]. Expression of HOXB7- and HER2-positive breast cancer induces JAK/STAT signaling, causing breast cancer cells to proliferate and metastasize [86]. The BK channel plays an important role in regulating the proliferation of HER2-positive breast cancer cells. In HER2-positive breast cancer, the expressions of HER2 and EGFR activate cyclin's high expression through Akt and STAT3 [87]. In TSC2-deficient HER2-positive breast cancer cells, the EGFR-STAT3/CD24 loop specifically activates breast cancer cells to proliferate [88]. STAT3 interacts with GLI1 AND tGLI1 to activate the transcription of the proliferation-related genes R-Ras2, Cep70, and UPF3A, thereby enhancing the tumorigenesis and spheroidization ability of HER2-positive breast cancer cells [89]. IL-30 expression was higher in HER2-positive breast cancer, which was associated with breast cancer recurrence. IL-30 promotes the proliferation of HER2-positive breast cancer cells through KISS1 and STAT1/STAT3 signaling [90]. The activation of JAK2/STAT3 signaling in HER2-positive breast cancer cells affects the activation of the p38 MAPK signaling pathway and promotes breast cancer cell proliferation [91]. Dihydrofolate reductase (DHFR), an important downstream mediator of HER2 signaling, promotes breast cancer cell proliferation through the Src/STAT3 pathway [92]. Myeloid-derived suppressor cells (MDSCs) and the less suppressive granulocyte (G)-MDSC phenotype are critical components of cell proliferation in HER2-overexpressing breast cancer cells. Tumor-associated macrophages also epigenetically reprogram the phenotypes of M1 and M2 macrophages to improve the immunosuppression of tumor cells [93]. The phosphorylation of JAK1/STAT3 is critical for the proliferation of HER2-positive breast cancer cells. HER2 activation increases the nuclear translocation of STAT3 through Jak1 to promote cell proliferation [94]. FA may lead to the occurrence and progression of breast cancer. The presence of FA increases the expression of several proteins, such as HER2 and STAT3, as well as various growth and inflammation-related factors, thereby causing tumor cells to maintain a high proliferation level [95]. IL-6 is the activated form of STAT3, a crucial transcription factor downstream of HER2. Overexpression of HER2 can produce high levels of IL-6, which activate STAT3 and stimulate cell proliferation [96]. When HER2-positive breast cancer cell lines develop brain metastasis, M2 microglia infiltrate massively. At this time, tumor cells in the brain metastasis microenvironment of breast cancer in vivo will secrete many chemokine ligands of the C–C motif, which will enhance the stemness of tumor cells. Estrogen in the microenvironment increases signal regulatory proteins on microgliaα and limits it's phagocytic capacity, allowing for high-level proliferation [97]. In the MCF7 breast cancer cell line, which overexpresses both ER and HER2, HER2 activation can promote the HER2/MAPK/ERK pathway to activate proliferation and metastasis. However, many invasion and metastasis proteins, including STAT3 (CREB, STAT3, cancer stem cells (CSCs), Fak, Pax, and Fascin), exhibit abnormal expression under the dual influence of ER and HER2 [98]. Kinase array analysis of HER2-overexpressing breast cancer cell lines revealed that the expression levels of caspase-8, caspase-3, and PARP1 were decreased, while phosphorylated STAT3^Y705 was upregulated and phosphorylated p21^T145 was downregulated. This result demonstrates that HER2 overexpression promotes DNA damage repair through STAT3 [99]. Another transcriptomic and proteomic study of HER2-positive breast cancer cells identified that the PIK3CA^H1047R mutation promoted the proliferation and vascularization of breast cancer cells through the STAT3 and VEGF/HIF signaling pathways. Overexpression of both HER2 and ER, ER and PI3KCA^H1047R in luminal B breast cancer cells can increase the expression level of YAP_pS127 through the STAT3, MAPK, Akt, and Hippo pathways, enabling breast cancer cells to acquire resistance to Bcl-2 family and MEK/MAPK inhibitors [100]. EDI3 was found to be highly expressed in HER2-positive breast cancer cells, and it's overexpression has been associated with PI3K/Akt/mTOR and GSK3β downstream pathways, as well as transcription factors (HIF1α, CREB, and STAT3) [101]. TNF-α with IFN-γ increases STAT1 phosphorylation through serine and tyrosine sites and compensatory reduction of STAT3 activation to inhibit the high proliferation level of HER2-positive breast cancer cells. When EGFR or HER2 inhibitors are combined with TNF-α, they can also induce tumor cells and limit proliferation by inhibiting STAT3 [102].

However, some studies found that STAT3 inhibits the proliferation of HER2-positive breast cancer cells through specific mechanisms. After activation, HER2 or HER3 receptors promote signal proliferation in HER2-positive breast cancer cells through downstream signaling such as ERK and Akt. The specific pruning ability of the canonical p-Y705 or non-canonical p-S727 PTM of STAT3 protein can specifically modify HER2 and HER3 and inhibit the proliferation of breast cancer cells to a certain extent [103].

Sustained STAT3 activation is a hallmark of resistance to trastuzumab therapy. Crosstalk between HER2 and β2-AR affects the biological behavior of breast cancer cells. Catecholamine-induced β2-AR expression can antagonize the antiproliferative effect of trastuzumab. Catecholamines activate the HER2/STAT3 signaling pathway and inhibit miR-199a/b-3p, promoting the activation of HER2/PI3K/Akt/mTOR [104]. Trastuzumab resistance in HER2-positive breast cancer cells is attributed to the highly up-regulated expression of non-structural maintenance of chromosomal condensin 1 complex subunit G (NCAPG). NCAPG triggers a series of proliferation-related biological cascades by phosphorylating Src and by enhancing the nuclear localization and activation of STAT3 [105]. Nectin-4 interacts with HER2 to promote the production of ErbB2 spliceosome through the JAK/STAT3 signaling pathway and PI3K, leading to trastuzumab treatment failure, which is responsible for the continued high proliferation of breast cancer cells [106]. Sustained activation of STAT3 drives downstream HIF-1α/HES-1 continuous activation of the pathway renders trastuzumab therapy ineffective [107]. Another study of lapatinib-resistant HER2-positive breast cancer cells found that the level of IL-6 was significantly higher than that of the parental cell line and had a higher number and characteristics of stem cells. This is mainly because IL-6 can activate STAT3 [108]. In the same study of immune-deficient mice implanted with xenografts derived from breast cancer patients, the expression level of IL-6 was closely related to the phosphorylation level of STAT3, which was also closely associated with resistance to anti-HER2 therapy [109]. PDGFRA exhibited high expression levels in trastuzumab-resistant HER2-positive breast cancer patients. PDGFR can also induce the expression of IL-6 through the ligand PDGFC and increase the phosphorylation of STAT3 and ERK to stimulate cell proliferation and assist breast cancer cells in acquiring resistance to trastuzumab. Therefore, the PDGFRA/STAT3/IL-6 axis is closely related to the resistance to trastuzumab [110]. Smo and JAK2, as essential components of the trastuzumab-resistance in HER2-positive breast cancer cells, can activate VEGF-A expression through STAT3 to promote cell proliferation when trastuzumab inhibits HER2 [111]. Compared with normal HER2-positive breast cancer cells, the phosphorylation pathway of some key proteins of HER2 signaling (Akt, ERK, STAT3) and FOXP1 of trastuzumab-resistant SKBR3 cells exhibit high expression levels, and the cell cycle is also in a high proliferation cycle [112]. In the anti-HER2 therapy-resistant population, abnormal expression of anti-apoptotic proteins Bcl-2 and BIRC8 and other cell signaling proteins (Akt1, MAPK7, and RPS6KA5) is the key to drug resistance. The abnormal expression of these proteins is closely related to STAT3 [113]. EDI3 is also highly expressed in breast cancer cell lines that are resistant to anti-HER2 therapy [101].

STAT3, an important downstream signaling molecule of HER2, is also activated and transcribed due to the high expression of STAT3 when HER2 is blocked. Therefore, STAT3 promotes resistance of HER2-positive breast cancer to lapatinib [114]. When STAT3 is continuously activated, breast cancer cells will be endowed with resistance to EGFR TKIs, and the phosphorylation level of STAT3 and the activation level of FGFR/STAT3 will be increased at the same time [115]. Overexpression of the leukemia inhibitory factor receptor (LIFR) promotes the activation of STAT3, secretion of proliferation-related cytokines by STAT3, and induces breast cancer cells to acquire resistance to T-DMI [116]. CDK8/19 is a cyclin complex that is equally significant as CDK4/6. High CDK8/19 expression mediates STAT1 and STAT3 phosphorylation through the PI3K/Akt/mTOR pathway and upregulates the tumor suppressor BTG2 to promote the resistance of HER2-positive breast cancer cells to lapatinib [117]. The gene expression characteristics of CAFs are closely related to the clinical outcomes of patients receiving lapatinib. The expression levels of JAK2/STAT3 and hyaluronic acid were lower in the CAFs of lapatinib-sensitive patients. Lapatinib resistance leads to a higher expression of phosphorylated STAT3 and promotes the proliferation of breast cancer cells through the spatial proximity of Ki-67 and CAFs [118]. EGFR⁺/HER2⁺ patients mainly activate Src/STAT3/ERK1/2 signaling through IL-8 secreted by TAMs in the tme to interfere with the antiproliferative effect of lapatinib, causing cells to acquire resistance to lapatinib [119]. The upregulation of TG2 can be mediated by NF-κB signaling, which activates pathways leading to IL-6 upregulation in metastatic cells. This autocrine expression of IL-6 maintains enhanced TG2 levels through JAK/STAT3 signaling. This positive feedback pathway can continuously maintain the resistance level of neratinib [120].

STAT3 activation is closely related to breast cancer cells and the tumor microenvironment of HER2-positive breast cancer cells. CAFs, an essential component of the TME, appear during anti-HER2 treatment (trastuzumab and pertuzumab) and are expressed at high levels, along with the secretion of corresponding substances to reduce the sensitivity of cells to anti-HER2 treatment. According to LC–MS/MS quantification and miRNA analysis of breast cancer drug-resistant cell lines, STAT3 is also an important drug-resistance marker gene [121].

Radiotherapy, as one of the important methods for treating HER2-positive breast cancer in the clinic, has been proven to have a good therapeutic effect in breast cancer patients. However, radiotherapy resistance has also seriously affected the curative impact of breast cancer patients to a certain extent. HER2/STAT3/survivin regulation is associated with radiotherapy resistance in HER2-positive breast cancer. Increased expression of phosphorylated STAT3, STAT3, and survivin weakens the sensitivity of HER2-positive breast cancer cells to radiotherapy while increasing the incidence and severity of radiotherapy-related adverse reactions. This is mainly due to the involvement of the STAT3/survivin pathway in promoting the expression of PRAP, which facilitates cell repair of DNA damage caused by radiation. This is mainly due to the involvement of the STAT3/survivin pathway in promoting the expression of PRAP, which facilitates cell repair of DNA damage caused by radiation [122].

Currently, STAT3 inhibitors are widely used to inhibit the proliferation of HER2-positive breast cancer. STAT3-specific decoy oligonucleotides (STAT3 decoy ODNs) can block STAT3 signaling through CD44 mediated by creatine acid, inhibit the expression of downstream target genes, and inhibit the proliferation of STAT3-mediated HER2-positive breast cancer cells [123]. When using other protein inhibitors/agonists, STAT3 expression levels will also be correspondingly suppressed, reducing the high proliferation of HER2-positive breast cancer.

ER-positive breast cancer accounts for the largest proportion of breast cancer patients. ER-positive breast cancer cells can be stimulated by estrogen to activate proliferation signals and promote cell proliferation. The loss of AKTIP at 16q12.2 drives ERα. The occurrence of era-positive breast cancer is closely related to the poor prognosis of patients. Depletion of AKTIP increased era protein levels and activity. Cullin-associated and neddylation-dissociated protein 1 (CAND1) regulates cullin ring E3 ubiquitin ligase, which protects the era from cullin 2-dependent proteasomal degradation. In addition to era signaling, AKTIP loss triggers JAK2/STAT3 activation, providing an alternative survival signal when the era is inhibited [124]. Xist, a key regulator of breast CSCs, exhibits aldehyde dehydrogenase positive (ALDH⁺) epithelial-(E) and CD24^lowCD44^hi mesenchymal-like (M) phenotypes. Xist can stimulate the expression of the proinflammatory cytokines IL-6 and IL-8 in luminal breast cancer cells through microRNA let-7a-2-3p, thereby increasing the tumorigenesis and spheroidization ability of cells. Moreover, stimulating the expression of IL-6 to activate STAT3 promoted the activation and expression of CSC self-renewal key factors, including c-Myc, KLF4, and SOX9 [125]. Using chip qPCR and dual luciferase detection revealed that STAT3 can bind to the MID2 promoter fragment and increase MORC4 expression in luminal breast cancer. MORC4 can increase the expression level of Bcl-2 and cell proliferation [126]. PD-1 can promote cell invasion and metastasis in breast cancer cells, which depends on the integrity of PD-1 disability. In luminal breast cancer, PD-1-positive breast cancer cells activate STAT3 and STAT1 by releasing CXCL8. STAT1 and STAT3 help luminal breast cancer cells to increase proliferation through PD-1 mutants [127]. In a study of patients with luminal A- and HER2-positive tumors, it was found that the low expression level of PTPN2 protein was found to be closely related to a higher recurrence rate. After PTPN2 knockdown, met phosphorylation increases, ERK phosphorylation decreases, and EGF-mediated STAT3 activation increases, resulting in increased cell proliferation [128]. After analyzing clinical samples of breast cancer patients, it was found that the expression of TBC1D9 was negatively correlated with the proliferation index in luminal breast cancer patients. Low TBC1D9 expression alters the expression of proliferation-related genes in breast cancer cells (ARL8A, ARL8B, PLK1, HIF1α, STAT3, and SPP1), leading to increased cell proliferation [129]. Cancer-associated adipocytes (CAAs) in the microenvironment of luminal breast cancer can stimulate nearby tumor cells to increase proliferation by secreting increased levels of IL-8. Besides, IL-8 can enhance the carcinogenesis and proliferation of breast cancer in a STAT3-dependent manner [130]. Dats plays an important role in leptin-regulated oncogenic signaling in luminal breast cancer cells. Leptin promotes the proliferation of breast cancer cells by inducing STAT3 phosphorylation and nuclear translocation of breast cancer MCF-7 cells. The mRNA levels of proliferation-related cyclin D1 also exhibited high proliferation levels. After dats treatment, the level of phosphorylated STAT3 in mice and cell proliferation significantly increased [131]. CAFs in ER-positive breast cancer cells can stimulate BC cells to increase proliferation levels by secreting IL-6 in a STAT3- and p16-dependent manner [132]. Overexpression of the ER in ER-positive MCF-7 cells leads to high expression of proliferation genes (C-Ha-Ras, Rho-A, p53, CCND1), resulting in increased cell proliferation [133]. STAT1 and STAT3 complex promotes the proliferation of breast cancer cells. Somatic truncation and mutation of the prolactin receptor (PRLR) are necessary for developing ER-positive breast cancer cells. Truncated and wild-type PRLR lead to the activation downstream of STAT3 and STAT5 abnormal receptors, enabling cells to acquire high proliferation levels [134]. After using single-cell sequencing technology to detect luminal cells, it was found that tumor cells labeled with ITGB1/CD29 + and TGB3/CD61⁺ in ALDH⁺ breast cancer stem cells exhibited higher proliferation levels. However, breast cancer stem cells can pass through EGFR/STAT3 and TGFB/TGF-β/Smad exert autophagic effects to maintain stemness levels and high proliferation properties [135]. Luminal-type cells are more sensitive to proliferation and oxidative damage-induced senescence than their corresponding stromal fibroblasts. Senescent luminal cells can secrete inflammatory factors and express high levels of the p53 family, while LAMINB1 expression is downregulated. Senescent luminal breast cancer cells can also activate fibroblasts through the IL-8 pathway, which promotes the proliferation level of breast cancer cells [136]. When using DMBA and MPA-induced ER-positive breast cancer in mice, it was found that the expression level of AT1R was significantly higher in tumor tissues than in normal tissues. High AT1R expression promotes IL-6, p-STAT3, and TNF-α. Increased IL-6 expression causes tumor cells to proliferate more [137]. CD44⁺CD24 cells in luminal breast cancer cells possess stem cell-like characteristics and contribute to disease progression. CD44⁺CD24 breast cancer stem cells maintain high proliferation characteristics through JAK2/STAT3 signaling [138]. In the TME of breast cancer, stimulation of various hormones and growth factors enhances the high proliferation level of breast cancer cells. TME improves tumor cell proliferation by stimulating STAT3. It can also induce IL-8 and PD-L1 to protect cells against immune crosstalk while maintaining strong proliferation properties [139]. ALDH⁺ and CD29^hiCD61⁺ in luminal breast cancer cells exhibit more proliferative properties than normal tumor cells. Breast cancer stem cells with ALDH⁺ and CD29^hiCD61⁺ maintain high proliferation through STAT3 or TGF-β/Smad [140].

Although current estrogen antagonistic and CDK4/6 therapies for ER-positive breast cancer have demonstrated significant therapeutic benefits for luminal breast cancer patients, drug resistance will inevitably occur in some patients after a period of treatment because of the unique biological functions of breast cancer cells. In a luminal breast cancer model, CAFs can transfer miR-221 to tumor cells through microvesicles to acquire therapeutic resistance. The highly expressed IL6/p-STAT3 pathway promotes the expression of ONCO/miR-221^hi CAF microvesicles, enabling breast cancer cells to pass ERs by improving treatment resistance α Maintain high proliferation levels [141]. When tamoxifen resistance develops in ER-positive breast cancer cell lines, the estrogen expression level decreases, and that of EGFR increases. EGFR enhances tamoxifen resistance, promotes Cip1 expression in the nucleus through the EGFR/Src/STAT3 signaling axis, and maintains a high level of cell proliferation [142]. In luminal breast cancer, abnormal FN expression is strongly associated with a poor prognosis of patients, which is primarily due to FN-mediated tamoxifen resistance. In tamoxifen-resistant breast cancer cells, FN promotes the high proliferation level of cells through the PI3K/Akt/STAT3 pathway [143]. Adhesive properties of endothelial cells (ECs) significantly affect the proliferation level of breast cancer cells. ECs activate the STAT3 and VEGFR2 pathways by secreting proinflammatory cytokines (IL-6 and IL-8) and angiogenic factors (VGFR) to stimulate the proliferation of breast cancer cells [144].

Although HER2-positive and ER-positive breast cancers can promote the proliferation of breast cancer cells by increasing STAT3 expression through a variety of signaling pathways, a variety of inhibitors, including STAT3 inhibitors, can still be used to inhibit the high proliferation level of breast cancer cells. Although various targeted therapeutic drugs can effectively improve curative effects and prolong the survival of patients with breast cancer harboring positive growth-promoting, drug resistance will inevitably develop. STAT3, an important cause of resistance to HER2 and ER inhibitors, can effectively reverse drug resistance using STAT3 inhibitors.

STAT3 is closely linked to the level of apoptosis

The anti-apoptotic property is a property of all tumor cells. Tumor cells can immortalize breast cancer cells by inhibiting the expression of Pan apoptosis-related signaling pathways. STAT3 promotes cell survival and inhibits apoptosis by regulating apoptosis-related proteins. The Bcl-2 and caspase families, essential regulators of programed cell death, can regulate programmed cell death through protein expression level [145]. Low expression levels of pro-apoptotic proteins and high expression levels of pro-survival proteins in HER2-positive and luminal-positive breast cancer cells result in the continuous occurrence and development of breast cancer and treatment resistance [146]. STAT3 can achieve the anti-apoptotic ability of breast cancer cells by regulating the expression levels of the Bcl-2 and caspase families [147]. Blocking STAT3-mediated gene regulation inhibits the expression of pro-survival proteins to induce apoptosis in breast cancer cells.

HER2 inhibitors and/or inhibitors of additional STAT3 upstream targets can inhibit the high proliferation level of breast cancer cells and restore the high expression level of pro-apoptotic factors. HER2 inhibitors can suppress JNK, STAT5/ERK/MAPK, STAT3, CREB, and p70 S6 kinase phosphorylation/activation of IKBA and p38 to promote apoptosis in HER2-overexpressing breast cancer cells [85]. Pyrroltinib, a pan-target EGFR family inhibitor, can also specifically target TSC2-deficient cells through the EGFR/STAT3/CD24 loop to promote apoptosis [88]. Acetylshikonin inhibits HER2/Src/STAT3 and induces the activation of apoptosis in HER2-positive breast cancer cells through caspase-3 [92]. Treating HER2-positive breast cancer mice with entinostat and ICIs can reduce immunosuppression and increase antitumor response by inducing changes in multiple bone marrow cell types, thereby improving ICI sensitivity. The combined treatment inhibits STAT3 and enhances cell death by inducing immunogenic cell death in breast cancer cells [93]. Quercetin upregulates the levels of the cleaved proteins caspase-8 and caspase-3 through caspase-dependent exogenous apoptosis and induces the expression of PARP. Quercetin induces high-level expression of apoptotic signaling by inhibiting STAT3 at concentrations > 20 µM [94]. STAT3 decoy ODNs enhance trastuzumab-induced apoptosis. For trastuzumab-resistant breast cancer cells, ODNs enhance trastuzumab sensitivity and cytotoxicity and induce apoptosis [123]. Although ibrutinib treatment can activate caspase-8, the cleavage of caspase-3 and PARP1 changes nuclear morphology and causes apoptosis through the caspase-dependent extrinsic apoptosis pathway. Ibrutinib can also promote DNA damage and apoptosis by downregulating STAT3 expression [123].

Mutations and/or abnormal expression levels of other genes (miRNAs, lncRNAs, and so on) and proteins can also enable HER2-positive breast cancer cells to acquire anti-apoptotic effects through STAT3. Other genes and proteins can also induce apoptosis by affecting STAT3 expression levels. miR-124 regulates HER2 expression by directly targeting STAT3. After miR-124 overexpression, the cell death of HER2-positive breast cancer after irradiation will significantly increase [148]. HER2/STAT3/survivin, an important signaling pathway for the survival of HER2-positive breast cancer cells, increases the sensitivity of HER2-positive breast cancer cells to radiotherapy and enhances the degree of cell death due to radiation-induced DNA damage after inhibition [122]. SP induces apoptosis by increasing ROS levels and inhibiting p38 MAPK phosphorylation and JAK2/STAT3 signaling [91]. The cytotoxic effects of CAR-T and NK cells on HER2-positive breast cancer cells can be enhanced by inhibiting hydrogen sulfide synthase, cystathionine b-synthase, and cystathionine c-lyase through the miR-155/NOS2/NO signaling pathway [149]. FcR stimulation of NK cells induced by IL-21R, IFN secretion by tumor cells exposed to antibody coating was enhanced. After trastuzumab treatment of HER2 breast cancer cells, NK cells upregulate IL-21R and enhance ICD effects [150]. After PI3KCA^H1047R in her overexpressing cells, signal pathways such as STAT3 and VEGF/HIF were overexpressed. Elevated YAP1 mRNA expression is also caused by PI3KCA^H1047R, leading to the cells acquiring resistance to Bcl-2 family inhibitors [100].

As STAT3 expression levels are closely related to HER2 inhibitor resistance, abnormal STAT3 expression will also affect the ADCC effect triggered by trastuzumab. NCAPG is closely associated with trastuzumab resistance. NCAPG overexpression enhances the anti-apoptotic ability of HER2-positive breast cancer cells. After NCAPG silencing, the anti-apoptotic ability of HER2-positive breast cancer cells is weakened [105]. MPA in trastuzumab-resistant and trastuzumab-sensitive HER2-positive breast cancer cells can induce apoptosis of HER2-positive breast cancer cells by inducing the rise of apoptosis-related proteins and attenuating the phosphorylation levels of some key proteins (Akt, ERK, and STAT3,) of HER2 signaling that inhibit apoptosis [112]. Another study found that anti-apoptotic proteins (Bcl-2 and BIRC8) of trastuzumab-resistant patients also exhibited high expression levels, mediated by the high expression of STAT3. Silencing STAT3 or anti-apoptotic proteins can increase the apoptosis of breast cancer cells under the action of ruxolitinib (by targeting Akt), everolimus (by targeting EGFR, MAPK7, RPS6JA5, and HER2), and erlotinib (by silencing Bcl-2 and BIRC8) [113]. After TNF-α and IFN-γ treatment of trastuzumab-sensitive and -resistant HER2-overexpressing cells, the senescence and apoptosis of tumor cells increase synergistically. The intervention of TNF-α and IFN-γ increases STAT1 phosphorylation, Compensatory reduction in STAT3 activation [102].

STAT3 can also promote anti-apoptotic and acquired drug resistance in luminal breast cancer cells by regulating the expression level of anti-apoptotic factor family-related proteins. Similar to HER2-positive breast cancer cells, the use of STAT3 and upstream signaling pathway inhibitors can induce apoptosis in luminal breast cancer cells by inhibiting STAT3 expression. Senescent normal breast lumen (NBL) can activate STAT3 to activate fibroblasts by secreting large amounts of IL-8. High IL-8 levels can also induce the release of normal breast epithelial cells and stimulate the development of breast cancer [136]. TNFAIP (A20) activates PSTAT3-mediated inflammatory signaling to promote a strong emt/csc phenotype. After inhibiting A20, TNF-α induced apoptosis in luminal breast cancer cells [151]. After curcumin-intervened MCF-7 cells, IκBα and STAT3 gene expression were inhibited. The intervention of curcumin combined with paclitaxel increased the expression of p53, Bid, caspase-3, caspase-8, and Bax and decreased the expression of Bcl-xL. This confirmed that curcumin can promote breast cancer cell apoptosis through STAT3, thereby affecting apoptosis-related protein instrument [133]. MORC4 inhibition significantly reduced the enrichment of STAT3-bound MID2 promoter fragments. MORC4 overexpression significantly increased Bcl-2 expression in MCF-7 cells, increasing their resistance to ADM, 5-FU, and DDP. Inhibiting MID2 or STAT3 can reverse this resistance [126]. The STAT3 signaling pathway mediated by EGFR and Src is activated in tamoxifen-resistant luminal-type cells. The inhibition of STAT3 may be a potential target for tamoxifen-resistant breast cancer. An increase in nuclear p21 (Cip1) may be the step in TAMR cell death induced by key STAT3 inhibitors [142]. PIK3CA^mut LBC cells can recruit MDSCs while excluding cytotoxic T cells through the arachidonic acid (AA) metabolic pathway by inducing immunosuppressive time. Inhibition of PIK3CA^mut can promote the transformation of cold tumors into hot tumors through PI3K/5-Lox/LTB4 signaling, thereby enhancing the efficacy of immune checkpoint inhibitors in luminal breast cancer [152].

STAT3 effectively regulates the phenotype of breast cancer cells

Cell plasticity refers to the ability of cells to reprogram and change their fate and identity in response to internal or external factors. Plasticity is not limited to stem cell features. Cells can acquire different phenotypes through dedifferentiation, transdifferentiation, and epithelial-mesenchymal transition (EMT). The ability of differentiated cells to return to a stem cell-like state is important for tumorigenesis, and some oncogenic drivers affect plasticity during tumorigenesis [153].

CSCs express stem cell-like programs that self-renew, maintain tumor growth, and generate tumor cells with restricted proliferative capacity. In a strict sense, CSCs generate subpopulations with limited growth and differentiation potential and never return to the CSC state. There is evidence that both CSCs and non-CSCs are plastic and may undergo phenotypic transitions under certain conditions [154]. The CSC niche comprises heterogeneous and interacting cell populations that play a major role in tumorigenesis and are essential for CSCs to regulate and promote cancer cell plasticity. A vascular niche is a specialized, highly vascularized area composed of ECs, pericytes, smooth muscle cells, and immune cells that create a tumor-permissive microenvironment by affecting stemness, chemoresistance, invasion, and metastasis. Intervention of CSC-related plasticity-related genes through various methods, including drugs, has become a new method in the field of cancer treatment. STAT3, a signal transcription factor, can be phosphorylated under the stimulation of upstream signals and enter the nucleus to transcribe stem cell-related factors, thereby stimulating the transformation of breast cancer cell plasticity.

STAT3 and TrkA are significantly co-overexpressed and co-activated in HER2-positive breast cancer. STAT3 is a novel substrate of TrkA. β-NGF-mediated TrkA activation induces TrkA-STAT3 interaction and promotes the STAT3 target genes SOX2 and Myc expression. These pathways promote the transformation of breast cancer stem cells [155]. MEDI5117 (an IL-6 antibody) inhibits IL-6/STAT3 activation by targeting CD44⁺CD24⁺ breast cancer stem cells, exerts antitumor activity, possesses a strong antitumor effect on trastuzumab-resistant cells, and overcomes tumor resistance [156]. IL-6 expression is elevated in lapatinib-resistant HER2-positive breast cancer cells, and the number and characteristics of stem cells are maintained. This result demonstrated that in HER2-positive breast cancer cells, an increase of IL-6 is required to maintain stemness properties, primarily mediated by activating STAT3. Moreover, IL-6 activity can reduce the number of breast cancer stem cells, inhibit proliferation, and overcome drug resistance [108]. When trastuzumab resistance develops in HER2-positive breast cancer cells, JAK2 inhibitors (ruxolitinib and pacritinib) can synergize with SMO inhibitors (vismodegib and sonidegib) to reduce the stemness level of breast cancer stem cells, and jointly reduce the stemness level of breast cancer stem cells and overcome drug resistance by inhibiting STAT3 [111]. Xist, a key regulator of breast CSCs, is highly expressed in luminal breast cancer cells with ALDH + and CD24^loCD44^hi. Xist can maintain the stemness level of breast cancer stem cells by activating STAT3 through the transcription of IL-6 and IL-8. In contrast, STAT3 promotes the self-renewal of key CSC factors (c-Myc, KLF4, and Sox9), along with their activation and expression [125]. In MMTV PyMT breast tumors, ITGB1/CD29⁺ and TGB3/CD61⁺ markers enrich breast stem-like cells and identify luminal progenitor-like cells with ALDH⁺. These stem cell characteristics are maintained by macroautophagy/autophagy. Autophagy maintains breast cancer stem cell characteristics through EGFR/STAT3 and TGFB/TGF-β/Smad signaling pathways [135]. Similarly, IL-6 and IL-8 can stimulate fibroblast proliferation by activating the STAT3 pathway. Breast cancer stromal fibroblasts can continue to maintain the stemness expression of luminal breast cancer cells by secreting proinflammatory cytokines [136]. The use of anti-CD44 antibody-coated IL6R antibody can reduce the high metastatic characteristics of breast cancer stem cells and reduce the stemness level by inhibiting IL6R/STAT3 signaling, regulating TME and stemness-related gene (SOX2) [157]. Luminal breast cancer cells with CD44⁺CD24 have stem cell-like characteristics and contribute to disease progression. CD44⁺CD24 cells can acquire resistance to paclitaxel and doxorubicin through JAK2/STAT3 signaling [138]. CSCs in luminal a breast cancer cells also maintain their stemness due to TME stimulating STAT3 and p65 phosphorylation levels [139]. ALDH + and CD29^hiCD61⁺ luminal breast CSCs can maintain the stemness level of breast cancer stem cells through the STAT3 or TGF-β/Smad pathways, leading to stronger proliferation and invasion ability [140].

STAT3 regulates vascular growth in breast cancer

Angiogenesis in breast cancer is a highly complex process that generally includes the steps of vascular endothelial matrix degradation, migration, ECs proliferation, ECs vascularization, and branching to form a vascular ring and a new basement membrane [158]. Angiogenesis in breast cancer occurs when breast cancer cells release angiogenic factors that activate vascular ECs and promote their proliferation and migration. On the other hand, it also occurs because some vascular growth factors secreted by ECs stimulate tumor cell growth. Moreover, breast cancer cells can promote vasculogenic mimicry by activating angiogenesis-related targets, which helps breast cancer cells obtain the energy needed for proliferation and creates favorable conditions for invasion [159]. The interaction between breast cancer cells and ECs runs throughout the entire tumor angiogenesis process.

A series of angiogenic signaling factors are activated and expressed during angiogenesis in breast cancer. Various biomolecules that promote or inhibit angiogenesis constitute a complex and dynamic angiogenesis system, including growth factors (VEGF, FGF, transforming growth factor, HGF), adhesion factors (integrins and cadherins), proteases (matrix metalloproteinases), extracellular matrix proteins (fibronectin and collagen), transcription factors (hypoxia-inducible factor and nuclear factor), angiogenin, angiostatin, endostatin, and interleukins [160]. These molecules activate gene expression through transmembrane receptors and induce EC proliferation, survival, and angiogenesis. As representative angiogenic signaling pathways, VEGF/VEGFR, PDGF/PDGFR, FGF/FGFR, and other signaling pathways are highly expressed in HER2-positive and luminal breast cancer microenvironments [161]. STAT3, an essential transcription factor of the angiogenic factor family, can stimulate angiogenesis via transcription of angiogenic factors. The use of STAT3 inhibitors can inhibit angiogenesis.

After intravenous injection of IL-30 into breast cancer-bearing mice, IL-30 activates STAT1/STAT3 to promote proliferation and vascular dissemination of breast cancer cells and stimulates the generation of breast cancer-related blood vessels [90]. MEDI5117, an IL-6 antibody, inhibits IL-6/STAT3 activation, EC growth, and angiogenesis [156]. FA may lead to the occurrence and progression of breast cancer. Injecting FA into healthy mice results in abnormal mammary tissue development, high expression of breast cancer-related proteins (HER2, FR, CA15-3, VEGF, STAT3, IL-6, TNF-α, and IL-1β), and malignant mammary epithelial cells. Injecting FA into breast cancer-bearing mice activates STAT3 and transcribes VEGF to stimulate breast cancer-related angiogenesis [95]. When the PIK3CA^H1047R mutation occurs in HER2-positive cells, the expression levels of the STAT3 and VEGF/HIF signaling pathways change. EIF4G, a key differential protein caused by PIK3CA^H1047R mutation, promotes tumor angiogenesis by regulating the hypoxia activation switch of PIK3CA^H1047R mutated HER2-positive breast cancer cells [100]. When HER2-positive breast cancer cells develop trastuzumab resistance, they exhibit high levels of PDGFRA expression. Meanwhile, PDGFC, a ligand of PDGFR, induces IL-6 expression. PDGFR inhibitors (ponatinib and sunitinib) reduce IL-6 expression. This indicates that the PDGFRA/STAT3/IL-6 axis is closely linked to trastuzumab resistance. PDGFR can facilitate breast cancer cells to evade trastuzumab by stimulating vascular survival [110]. CAAs enhance the pro-angiogenic effects of breast adipocytes in a STAT3-dependent manner by secreting higher levels of IL-8. Using a specific shRNA, anti-IL-8 antibody, or rapamycin to inhibit IL-8 signaling inhibited CAAs to inhibit breast cancer-related angiogenesis [130].

STAT3 is an important factor in regulating invasion

Tumor cells have the characteristics of invasion and metastasis. More than 90% of breast cancer patients die from multiple organ failure caused by developing metastatic lesions in other organs. As most breast cancer patients undergo radical resections in the early stages, they are unable to survive in the primary part. Thus, most patients with advanced breast cancer have metastatic breast cancer. In luminal-type and HER2-positive breast cancer cells, tumor cells are located in cell membrane-related receptors, which can continuously promote the expression of proteins related to invasion and metastasis, thereby continuously promoting breast cancer progression. HER2-positive and ER-positive breast cancer cells can undergo invasive changes through STAT3 after HER2 and er are activated. Invasive changes in breast cancer cells mainly include cytoskeleton remodeling, secretion of invasive factors (MMP2 and MMP9), and so on [162].

HOXB7 expression is associated with poor prognosis in HER2-positive breast cancer patients. Overexpression of HOXB7 in the immortalized mouse mammary epithelial cell line nmumg promotes lung metastasis of breast cancer cells by activating JAK/STAT signaling [86]. The HER2/HER3 complex promotes WASF3 phosphorylation and transcriptional upregulation. This effect is mainly induced by activating JAK/STAT signaling by HER2/HER3. High WASF3 expression can improve the invasive ability of breast cancer cells and their invasion and metastasis [163]. IL-30 can also induce invasive migration and KiSS1-dependent metastasis through STAT1/STAT3 signaling [90]. RSK is the key signaling node of HER2/MAPK/ERK pathway activation. RSK, a critical protein in many signaling pathways linked to EMT induction and metastasis development, can enhance invasion and metastasis by regulating EMT-related proteins (CREB, STAT3, CSC, FAK, Pax, Fascin, and Actin) [98]. In HER2-positive breast cancer cells, CDK8/19 promotes changes in tumor stromal components by regulating the PI3K/Akt/mTOR pathway and STAT1/STAT3 phosphorylation levels, thereby facilitating tumor cell invasion [117]. The co-overexpression of STAT3 and GLI1/tGLI1 increased the invasiveness of breast cancer. The transcriptional binding sites of STAT3, GLI1/tGLI1, R-Ras2, Cep70, and UPF3A can have an important impact on the invasion of HER2-positive breast cancer cells [89].

In patients with luminal breast cancer, ER-positive breast cancer cells and various components of the TME can promote the invasion and metastasis of breast cancer cells by activating the STAT3 signaling pathway. Breast cancer cells of luminal breast cancer are more aggressive to short-term TRIEN exposure. This effect is primarily achieved through Akt kinase and STAT signal transduction [164]. IL-6/STAT3 signaling drives metastasis of ER-positive breast cancer. As STAT3 and ER enhancers share the same transcription sequence, IL-6 promotes high-level expression of STAT3 and ER and activates the transcription program of invasive factors by STAT3 [165]. TBC1D9 expression in luminal breast cancer cells limits tumor cell invasion, closely related to the expression of invasion-related proteins (ARL8A, ARL8B, PLK1, HIF-1α, STAT3, and SPP1) [151]. CAAs isolated from invasive breast cancer cells are proinflammatory and exhibit an active phenotype. Compared with adjacent tumor-corresponding adipocytes (TCA), CAAs possess higher proliferation, invasion, and migration abilities. This effect is mainly due to CAAs secreting high levels of IL-8 and enhancing the invasion ability of breast cancer cells in a STAT3-dependent manner [130]. Leptin induces STAT3 phosphorylation and nuclear translocation in MCF-7 cells, improving their migration and/or invasion ability. Leptin promotes breast cancer cell proliferation by inducing the phosphorylation and nuclear translocation of STAT3. This process begins with leptin binding to its receptor (Ob-R) on the surface of breast cancer cells, thereby activating the JAK/STAT signaling pathway. The leptin receptor (Ob-R) is a transmembrane receptor with tyrosine kinase activity, capable of activating associated JAKs. Upon formation of the leptin-Ob-R complex, JAK family tyrosine kinases are activated. Activated JAK then induces phosphorylation of STAT3 at a specific tyrosine residue (Tyr705). Once phosphorylated by JAK, STAT3 undergoes dimerization, with two phosphorylated STAT3 molecules associating to form a homodimer. Phosphorylation and dimerization of STAT3 result in conformational changes, exposing its NLS, which facilitates the translocation of the STAT3 dimer into the nucleus through the nuclear pore complex [131]. CAFs in breast cancer tissue can promote transformation from mesenchymal to epithelial cells in a STAT3- and p16-dependent manner via IL-6 secretion. CAFs in the tumor tissues of breast cancer patients express lower levels of N-cadherin and higher levels of vimentin [132]. Another study found that CAFs can transfer miR-221 to tumor cells through derived microbubbles and promote bone metastasis of tumor cells via the IL-6/p-STAT3 pathway [141]. AT1R expression promotes luminal breast cancer cell infiltration by promoting IL-6, p-STAT3, and TNF-α [137]. Single-cell sequencing of CD44 + CD24 luminal breast cancer stem cells revealed that p-STAT3 closely regulates EMT in paclitaxel-resistant cells [138]. "TME stimulation" (estrogen + TNF-α + EGF, which represents the three arms of TME) can change the EMT characteristics of luminal breast cancer cells. This is primarily because TME stimulation induces the activation of S727-STAT3, Y705-STAT3, STAT1, and p65 and promotes the expression of invasion-related proteins [139]. Adhesion ECs can stimulate the activation of STAT3 and VEGFR2 pathways by secreting IL-8, IL-6, and VEGF and promote the metastasis of breast cancer cells through NF-κB as the primary regulator of the adhesion phenotype. Stem cells in luminal breast cancer cells can exhibit stronger invasive and EMT abilities. This is mainly achieved through STAT3 or TGF-β/Smad [140].

STAT3 is an important intermediate link between inflammation and cancer.

Most DNA damage caused by inflammation is attributed to reactive oxygen and nitrogen species (RONS). RONS evolve from immune cells to destroy pathogens but can also damage nearby human cells. Both innate and acquired immunities can promote immune cell aggregation through inflammatory reactions, but these reactions can also lead to DNA damage. Many factors involved in DNA damage reactions are proinflammatory. DNA damage can also promote inflammation through cell death and aging. All these factors contribute to inflammation accompanied by tumors, and many products in the inflammatory environment (interleukins, lactic acid, and so on) can stimulate tumor cells to proliferate, invade, and undergo metabolic reprograming, among other reactions [166].

The microenvironment of tumor cells contains several interleukins. Many interleukins promote the proliferation and invasion of breast cancer cells by stimulating STAT3 activation. However, in HER2-positive and ER-positive breast cancers, STAT3 promotes malignant biological behavior changes in breast cancer cells and transcribes inflammatory factors via STAT3 due to the activation of cell membrane-related receptors (HER2 and ER). The extracellular interleukin family also stimulates cell proliferation and invasion by transcribing inflammatory factors (IL-6, IL-8, and so on) through STAT3 [26]. Therefore, interleukins can activate the positive feedback pathway formed by STAT3, which continuously promotes the proliferation and invasion of tumor cells and improves their tolerance to treatment.

Genome instability is the sign and premise of cancer, for which inflammation is an important cause. The major cause of DNA damage produced by inflammation is due to (RONS). RONS evolved from immune cells to destroy pathogens but can also damage nearby human cells. Both innate and acquired immunity can promote the aggregation of immune cells through inflammatory reactions, which can also lead to DNA damage. Many factors involved in DNA damage reactions are proinflammatory. DNA damage can also promote inflammation through cell death and aging. All these factors lead to inflammation accompanied by tumors, and many products in the inflammatory environment (interleukin, lactic acid, and so on) can stimulate tumor cells to proliferate, invade, undergo metabolic reprogramming, and other reactions [167].

HER2-positive breast cancer cells initiate and maintain the inflammatory environment necessary to promote tumorigenesis. Overexpression of HER2 activates the pre-feedback activation loop of IL-1α and IL-6, thus stimulating NF-κB and STAT3 pathways to produce and maintain breast CSCs [168]. IL-30 is highly expressed in HER2-positive breast cancer cells and promotes the proliferation and invasion of tumor cells through STAT1/STAT3 and KISS1-dependent signaling pathways [90]. FA promotes tumor cell proliferation, invasion, and angiogenesis by stimulating inflammatory factors (TNF-α, IL-6, and IL-1β) in HER2-positive breast cancer cells and reducing the expression of IL-2 in TME [95]. IL-6 is an activated form of the key downstream transcription factors STAT3 and HER2 receptors. High IL-6 levels activate STAT3 and stimulate cell proliferation [96]. After FcR stimulation, IL-21 receptor (IL-21R) expression is significantly upregulated. IL-21 can activate NK cells and stimulate tumor cells to activate STAT1/STAT3 expression, thereby improving the therapeutic effect of IL-21R-coated trastuzumab [150]. When HER2-positive breast cancer cells undergo brain metastasis, a large number of M2 microglia are infiltrated in breast-brain metastasis. However, when tamoxifen is used to treat or surgically remove the ovaries, estrogen signal conduction becomes polarized, and the secretion of C–C motif chemokine ligand 5 is reduced, further inhibiting STAT3 signaling and thus limiting brain metastasis [97]. Anti-IL-6 therapy and IL-6 receptor expression are closely related to STAT3 expression in HER2-positive breast cancer cells, which is also linked to the therapeutic efficacy of anti-HER2 therapy. Blocking IL-6 can improve the sensitivity of tumor cells to HER2 therapy by inhibiting STAT3 [109]. When HER2-positive breast cancer cells become resistant to trastuzumab, PDGFC, a PDGFR ligand, promotes high-level expression of IL-6. PDGFR inhibitors (ponatinib and sunitinib) and STAT3 inhibitors can inhibit IL-6 expression and reduce trastuzumab resistance by inhibiting the PDGFRA/STAT3/IL-6 axis [110]. Treatment with anti-HER2 Th1 cells, TNF-α, and IFN-γ can increase STAT1 phosphorylation while decreasing STAT3 activation. This effectively reverses trastuzumab resistance in breast cancer cells [102]. When rapatinib resistance occurs, TAMs in the TME activate the Src/STAT3/ERK signal axis by secreting a high expression level of IL-8. The EGFR/Src signal axis can also interfere with lapatinib through the Src/STAT3/ERK signal axis [119].

Xist is a conditional factor for breast CSCs. It can be used as a regulator of miR let-7a-2-3p to promote IL-6 and IL-8 expression in ALDH + breast cancer cells. This process also promotes CSC self-renewal by activating STAT3 [125]. Overexpression of A20 in luminal breast cancer cells can generate an inflammatory microenvironment composed of granulocyte MDSCs to activate the inflammatory signal required by p-STAT3 to promote a strong EMT and/or CSC phenotype [151]. CAAs enhance the proliferation, invasion, and vascular survival of breast cancer cells in a STAT3-dependent manner and in vivo by secreting higher levels of IL-8 [130]. Tumor-associated fibroblasts (CAFs) in breast cancer TME can induce breast cancer cells to promote EMT in a STAT3- and p16-dependent manner by secreting IL-6 [132]. CAFs can also enhance miR-221 metastasis to promote hormone therapy resistance in luminal breast cancer cells. miR-221 increases tumor cell metastasis and drug resistance through the IL-6/p-STAT3 pathway [141]. Normal breast epithelial cells are more sensitive to aging caused by proliferation and oxidative damage than their corresponding matrix fibroblasts. When breast epithelial cells age, they secrete several cytokines (IL-6 and IL-8) that activate the STAT3 signaling pathway and promote the development of luminal breast cancer [136]. Besides, IL-6/STAT3 is an important cellular signaling pathway mediating the communication between tumor and immune cells. TME can be regulated by IL-6/STAT3, and the metastasis of breast CSCs can be affected by the expression of breast cancer-related genes (STAT3, SOX2, VEGFA, MMP-9, and CD206) [157]. EC activates STAT3 and VEGFR2 pathways by secreting IL-8, IL-6, and VEGF and finally stimulates the adhesion change in tumor cells through NF-κB, leading to the metastasis of breast cancer cells [144].

STAT3 is also a regulator of important basic hallmarks of other tumors

Tumor energetics is a relatively recent discipline in cancer cell research, which mainly focuses on the metabolic changes and metabolites of cancer cells. Oxygen, an important component of metabolic regulation, is closely associated with changes in the metabolic phenotype. It is generally believed that aerobic oxidation is the primary metabolic mode of cells in the presence of sufficient oxygen, and anaerobic glycolysis is the main metabolic mode when there is insufficient oxygen [169]. Aerobic glycolysis is the main metabolic mode in tumor cells, regardless of oxygen sufficiency, and is also called the "Warburg effect." The typical manifestations of the "Warburg effect" include an increase in glucose decomposition rate and an incomplete TCA cycle (in the presence of fully functional mitochondria and sufficient oxygen). Tumor cells can promote TCA and increase the production of adenosine triphosphate (ATP) by converting pyruvate into lactic acid through aerobic glycolysis, providing sufficient raw materials for the proliferation and invasion of tumor cells [170]. Cancer biology and metabolism are unable to define the metabolic difference between tumors and normal cells. However, some recent studies have confirmed that STAT3 is important in regulating the Warburg effect. According to certain research, STAT3 activation is closely related to the maintenance of PKM2/HIF-1α positive feedback loop.

TGF-mediated EMT is mainly realized through the non-classical Akt/STAT3 signal axis. Metabonomics found that breast cancer cells with TGF-mediated EMT (SK-BR-3 and T47D) exhibited a generally higher glycolysis level than breast cancer cells without EMT [164]. However, the Akt/STAT3 signal axis of HER2-negative breast cancer cells is activated after TRIEN stimulation, resulting in energy metabolism changes, with lipid metabolism being the main change [165].

Cancer immunology considers that the immune system can prevent the occurrence of tumors, and abnormal immune function is a primary cause of tumors. The immune system of normal people detects and destroys normal cells with replication errors. However, when the immune system is defective, it cannot accurately identify cells with incorrect replication, creating conditions for the formation of tumor cells [171]. Even when many immune cells infiltrate tumor tissues, proteins such as PD-1 and CTLA-4 evolved by tumor cells can escape the surveillance of the immune system, leading immune cells to mistake tumor cells for normal cells and allow tumor cells to proliferate. Immune escape mechanisms have been validated in laboratory and clinical research. Tumor immunotherapy methods are divided into two categories: immune cell therapy and cytokine therapy. Given the numerous drawbacks of immune cell therapy, cytokine therapy has recently emerged as a hot spot in tumor immunotherapy. STAT3, a regulatory factor involved in the expression of several immune-related proteins in immune and tumor cells, can prevent immune cells from accurately recognizing and attacking tumor cells, thereby allowing immune escape of tumor cells.

Overexpression of the HER2, MDSC, and G-MDSC phenotypes in breast cancer cells promotes the immune escape of tumor cells through NF-κb and STAT3 pathways. MDSCs and G-MDSCs also promote M1-like macrophages to become M2-like macrophages to promote the immune escape of tumor cells [93]. FcR stimulation upregulates IL-21R expression in NK cells of the breast cancer microenvironment. However, treating FcR-stimulated NK cells results in changes in the expression and phosphorylation levels of STAT1 and STAT3 in breast cancer cells. After FcR stimulation, activating NK cells with IL-21 can improve and mediate the ADCC effect of trastuzumab [150]. Treatment of HER2-positive breast cancer cells with CD4⁺ Th1 cells or Th1 cytokines TNF-α and IFN-γ in a dose-dependent manner causes aging or apoptosis. Cells sensitive to and resistant to trastuzumab also experience aging or apoptosis when the same intervention method is used, mainly due to increased STAT1 phosphorylation and compensatory decrease of STAT3 activation [102]. Overexpression of A20 in luminal breast cancer cells can create an inflammatory microenvironment composed of granulocyte MDSCs that promote the invasive metastasis of tumor cells [151]. PD-L1 activates STAT3 and STAT1 in tumor cells by releasing CXCL8, which changes the glycosylation level of PD-L1 and promotes cell proliferation, invasion, and immune escape [127]. When PIK3CA^mut develops in breast cancer cells, it induces immunosuppressive MDSCs and inhibits cytotoxic T cells from attacking breast cancer cells via AA metabolism. On the other hand, PIK3CA^mut activates the transcription-dependent mode of 5-LOX in STAT3, which increases the yield of LTB4 and promotes its combination with MDSCs, boosts the infiltration of MDSCs into tumor tissues, and makes breast cancer cells have stronger immunosuppression [152].

The acquisition of several malignant biological and behavioral functions of tumor cells depends on a series of changes in their genome [172]. Some mutant tumor genes can enrich tumor cells with a selective advantage, allowing them to grow in the local tissue environment and finally take advantage. Tumor development can be considered a process of gradual accumulation of a series of mutations. The accumulation of numerous mutations promotes the biological behavior of normal cells to change and evolve into cancer cells. Due to the genome maintenance system in the human body, the rate of spontaneous mutations in each cell generation is typically low. Therefore, tumor cells must continuously increase their mutation rate during tumor incidence and development to cope with the risks associated with wireless proliferation [173]. In breast cancer cells, mutations in key genes cause mutations and/or abnormal expression of STAT3, allowing tumor cells to maintain other malignant biological behavior functions.

PIK3CA^H1047R selectively alters STAT3 and VEGF/HIF in HER2-positive breast cancer. EIF4G is the key protein that PIK3CA^H1047R causes changes in HER2-positive breast cancer. An increase in eIF4G can regulate HIF-1 expression in HER2 PIK3CA^H1047R breast cells to promote tumor angiogenesis and tumor cell proliferation. The PIK3CA^H1047R mutation in ER-positive breast cancer cells promotes high-level expression of YAP_pS127 while simultaneously leading to drug resistance to Bcl-2 family inhibitors [100]. Loss in AKTIP on 16q12.2 drives the poor prognosis in ER-positive breast cancer patients. AKTIP-depleted tumors increase the level and activity of ERα protein. CAND1 regulates cullin RING E3 ubiquitin ligase and protects ERα from cullin 2-dependent proteasome degradation. The loss of AKTIP also triggers the activation of JAK2-STAT3, thus providing an alternative survival signal when ERα is suppressed [124]. In patients with ER-positive breast cancer, PD-L1 increases metastasis through the N-linked glycosylation sites (N35, N192, N200, and N219) of specific mutants. The N219, an important site for metastasis promotion, can be transferred by regulating the activation of STAT3 and STAT1 [127]. The somatic truncated mutant prolactin receptor (PRLR) provides a crucial environment for the proliferation of ER-positive breast cancer cells. However, both truncated and wild-type PRLR promote abnormal activation of the STAT3 and STAT5 signaling pathways and the occurrence of tumors [134]. The PIK3CA^mut induces ER-positive breast cancer cells to exhibit stronger immunosuppression by activating STAT3 [152].

Epigenetics describes gene expression changes that do not involve DNA sequence changes. Non-mutation epigenetic reprogramming is a critical link in cancer development, while genomic instability and favorable characteristics of mutation are the basic components of cancer development and pathogenesis. Non-mutant epigenetic reprogramming is an evidently independent genome reprogramming model that involves only epigenetic regulatory changes in gene expression [174]. Cancer cells can regulate gene expression in various ways, including regulating non-coding RNA, changing the chromatin state, and removing epigenetic modifications. Several studies have found that the composition of normal and tumor cells is approximately identical to that of the embryonic cells [175]. In normal adults, specific gene modifications, such as the modifications of genes and histones, the changes of chromatin structure, and the activation or inhibition of specific genes through complex feedback loops, can maintain gene expression levels. In tumor cells, abnormal regulatory switches activate multiple cancer proteins while inhibiting tumor suppressor proteins [176]. Non-mutant epigenetic reprogramming methods mainly include chromatin remodeling complexes, histone modifications, non-coding RNA, and other epigenetic mechanisms [177]. Epigenetic reprogramming of non-mutant breast cancer cells is not limited to breast cancer cells themselves. However, it includes three important aspects: epigenetic regulation of stromal cell types in the breast cancer cell microenvironment, epigenetic regulation of breast cancer cell heterogeneity, and epigenetic regulation of the breast cancer cell microenvironment. STAT3, a transcription factor, may induce the production of several non-coding RNAs through certain shearing modifications while transcribing the corresponding target protein. These non-coding RNAs affect the expression level of cancer protein/cancer-promoting protein. STAT3 expression is also regulated by epigenetic reprograming.

Catecholamines can activate HER2/STAT3, and they can also inhibit the promotion of HER2/PI3K/AKT/mTOR by miR-199a/b-3p, enabling HER2-positive breast cancer cells to acquire the ability to antagonize trastuzumab [104]. Nectin-4 cis-binds to ErbB2, activates the PI3K and JAK/STAT3 pathways, promotes DNA synthesis, and endows breast cancer cells with the ability to resist trastuzumab [106]. Overexpression of miR-124 significantly reduced the activity of STAT3 signaling pathway in HER2-positive breast cancer and significantly enhanced cell death after irradiation [148]. NCAPG was found to be ectopic and expressed at high levels in HER2-positive breast cancer cells that were trastuzumab-resistant. Overexpression of NCAPG enhances the nuclear localization and activation of STAT3 through Src phosphorylation, promoting proliferation and anti-apoptosis of breast cancer cells [105]. IL-6/STAT3 signaling drives metastasis of ER-positive breast cancer. As ER shares an enhancer with STAT3, activation of the IL-6/STAT3 signaling pathway is difficult to reach, the therapeutic level for standard ER-targeted therapies [165]. Breast cancer-associated CAFs promote the occurrence of onco-miR-221^hi vesicles and their metastasis to breast cancer cells by activating the IL-6/STAT3 signal axis. High-level expression of miR-221 can make luminal breast cancer cells obtain the effect of hormone therapy resistance [141]. miR-21-3p, miR-21-5p, and miR-221 also participate in anti-HER2 drug resistance by regulating STAT3 [117].

Cell senescence is a common, irreversible form of normal cell proliferation arrest. Cell senescence may be a potential protective mechanism for maintaining tissue homeostasis and a supplementary mechanism for programmed cell death, which inactivates and removes sick, dysfunctional, or unnecessary cells over time [178]. Cell senescence closes the cell division cycle and alters cell morphology and metabolism. Aging cells can stimulate tumor development and malignant progression in various ways. Generally, tumor cells do not age due to characteristics of telomerase [179]. However, cell and genome damage caused by chemotherapy, radiotherapy, and treatment-induced aging is also manifested in tumor cells. The characteristics of tumor cells that are difficult to age can also help tumor cells gain resistance to various treatments [180]. As a critical protein for proliferation and invasion, high levels of STAT3 expression in growth-promoting receptor-positive breast cancer cells maintain high proliferation characteristics and avoid aging.

Treatment with CD4⁺Th1 cells or Th1 cytokines TNF-α and IFN-γ in a dose-dependent manner causes breast cancer cells with high and moderate HER2 expression to undergo apoptosis and cell aging. This is mainly because TNF-α and IFN-γ increase the phosphorylation of STAT1 through serine and tyrosine sites, resulting in the compensatory decrease of STAT3 activation [102]. Active fibroblasts related to aging differentiate and proliferate, and primary human cells transform into pluripotent stem cells in an IL-8-dependent manner, promoting the development of ER-positive breast cancer. IL-8-related cavity cell dedifferentiation is induced by p16INK4A and STAT3 of miR-141 [181]. Aging NBL cells secrete many cytokines, including IL-6 and IL-8, and express high levels of p16, p21, and p53, whereas lamin B1 is down-regulated. During aging, cavity cells activate matrix fibroblasts in an IL-8-dependent manner by activating the STAT3 pathway. However, the role of aging mammary gland cells in promoting the inflammatory/carcinogenic microenvironment by activating fibroblasts in an IL-8-dependent manner [136].

Targeting STAT3 is a brand-new treatment for growth-promoting receptor-positive breast cancer

Due to its unique structural biological properties, STAT3 is expected to play its biological role through phosphorylation or combining to form dimers. The SH2 domain recognizes and binds to the receptor and other specific phosphorylated rosin residues on the protein, forming a polyprotein complex and mediating intracellular protein–protein interaction. Currently, there are two methods to directly inhibit STAT3 by targeting SH2: blocking the recruitment of activated RTK or non-receptor kinase (phosphorylating Tyr705 of STAT3) to the plasma membrane and blocking the dimerization of two activated STAT3 molecules [182].

Currently, both types of inhibitors (peptides and small molecules targeting the STAT3-SH2 domain) can inhibit tumor cell proliferation and induce apoptosis to some extent. Some peptidomimetic inhibitors are designed by amino acid residues based on the STAT3 structure. These inhibitors directly interact with the SH2 domain to inhibit dimerization. Both peptidomimetic and non-peptide small molecule inhibitors can restrict the proliferation of cancer cells and promote the accumulation of STAT3 in mitochondria to a certain degree, leading to mitochondrial dysfunction, ROS induction, and eventually cancer cell death [183]. It has recently been revealed that small molecules found in many natural drugs can also inhibit the SH2 domain of STAT3 [184]. Targeting the SH2 domain of STAT3 may lead to non-specific inhibition of proteins containing other SH2 domains, thereby affecting the activity of multiple signaling pathways. Many signaling pathways interact through SH2 domains. If the inhibition of STAT3 impacts these pathways, it may alter cellular responses to external signals. Given that SH2 domains are involved in various cellular processes such as proliferation, apoptosis, and immune responses, the inhibition of STAT3 could lead to unintended side effects, such as diminished immune function or abnormal cell growth. In the context of cancer or other disease treatments, if the suppression of STAT3 disrupts the balance of other signaling pathways, it may contribute to the development of resistance. Therefore, when targeting the SH2 domain of STAT3, it is essential to consider the potential impact on other SH2 domains and closely monitor the occurrence of side effects in clinical studies to ensure the safety and efficacy of the treatment [185].

The role of activated STAT3 in promoting proliferation, migration, and invasion of breast cancer cells depends on the physical interaction between the DBD and DNA after translocation into the nucleus. Inhibitors targeting the STAT3 DBD may also reduce STAT3 activity by destroying the binding of STAT3 to target DNA. However, STAT3 is an important transcription factor in cells, and its binding domain in DNA is considered to be difficult to measure. However, using a platinum-containing compound [186] and a small molecular lead compound [187], the transcription characteristics of STAT3 can be inhibited by specifically blocking the binding of STAT3 to DNA.

Reducing STAT3 mRNA levels can also reduce STAT3 expression. Antisense oligonucleotides can selectively inhibit STAT3 mRNA translation and reduce STAT3 expression through complementary binding with single-stranded RNA sequences. We have also confirmed this view through some basic research [188]. The stability of STAT3 antisense oligonucleotides can be effectively enhanced through methods such as chemical modification, liposomal or nanoparticle carriers, covalent attachment of protective groups, and conjugation with carrier proteins, thereby improving their therapeutic potential [189].

As a brand-new Proteolysis-Targeting Chimeras (PROTAC), PROTAC can reduce the protein expression level by specifically targeting protein hydrolysis. The principle of PROTAC technology is mainly based on the ubiquitin–proteasome system (UPS) in cells, which is a natural mechanism responsible for removing secondary proteins. The UPS system is catalyzed by E3 ligase, but it requires various proteins to obtain the substrate and identify the proteins that need to be degraded. PROTAC technology can induce the degradation of a target protein that does not require degradation by connecting the ligand of the target protein with the ligand, which helps UPS find the substrate protein through chemical bonds. This technology uses the ubiquitination process, in which ubiquitin molecules combine with the target protein to form a signal recognized by the proteasome, eventually leading to the degradation of the target protein. PROTAC is a heterobifunctional molecule composed of two ligands connected by a linker. One ligand binds to the target protein, whereas the other targets the E3 ligase. This structure enables PROTAC to bind the target protein and E3 ligase simultaneously, thereby shortening the distance between them and inducing ubiquitination of the target protein, allowing it to be recognized and degraded by the 26S proteasome. This process has high selectivity because it does not require high-affinity binding of PROTAC to the target or the specific active pocket of the target protein. In previous studies, STAT3 was considered a difficult target for drug development. PROTAC technology can be used to specifically perform ubiquitination modification on the STAT3 protein, reducing the purpose of its overexpression [190].

STAT3 decoy ODNs are a type of STAT3 inhibitor currently under investigation. Although they have shown promising anticancer potential in certain preclinical studies, there are significant challenges and limitations in their practical application. One of the primary issues is that oligonucleotides are prone to degradation by nucleases in vivo, especially in the bloodstream and tissues. This degradation substantially reduces the half-life of decoy oligonucleotides, thereby weakening their therapeutic efficacy. For effective inhibition, oligonucleotides need to be precisely delivered to tumor sites, but achieving specific targeted delivery in the human body is challenging. Non-specific uptake in healthy tissues can lead to off-target effects, increasing the risk of side effects. Furthermore, oligonucleotides are large molecules with a negatively charged phosphate backbone, making it difficult for them to cross cell membranes unaided. Additionally, STAT3 decoy oligonucleotides may non-specifically bind to other transcription factors or DNA-binding proteins with similar sequences, leading to off-target effects. These off-target interactions can interfere with the expression of other genes or signaling pathways, potentially causing adverse reactions. Oligonucleotides can also exhibit varying levels of toxicity depending on dosage, necessitating precise dosing to avoid harmful side effects. Prolonged administration may lead to cumulative toxicity, negatively impacting patient health. Moreover, STAT3 is often involved in crosstalk with other signaling pathways, such as PI3K/AKT signaling pathway and MAPK signaling pathway. Therefore, inhibiting STAT3 could indirectly affect these pathways, producing complex biological effects and even compensatory activation that could result in tumor resistance to treatment. While STAT3 decoy ODNs theoretically offer the potential to inhibit STAT3 activity, their clinical application faces multiple obstacles. These challenges significantly limit the further exploration of STAT3 decoy ODNs in both basic and clinical research settings [191].

STAT3 inhibitors exhibit great potential in clinical research on cancer treatment

So far, the US Food and Drug Administration (FDA) has approved several STAT3 pathway inhibitors for clinical cancer treatment. However, alternative STAT3 pathway inhibitors have been used in clinical trials. In clinical trials, the safety and antitumor effects of drugs such as AZD9150, HJC0152, BBI608, and SD-36 in cancer treatment have been evaluated. The first phase 0 experimental study of a STAT3 inhibitor used in humans (NCT00696176) revealed that no patients experienced grade 3/4 or dose-limiting toxicity. The levels of STAT3 target genes (cyclin D1 and Bcl-XL) were significantly decreased, and the tumor proliferation level of patients was inhibited [192]. AZD9150 is an antisense oligonucleotide targeting STAT3 with potential antitumor activity. AZD9150 binds to STAT3 mRNA, inhibiting the translation of transcripts and reducing the protein expression level of STAT3. A phase I dose escalation clinical trial including 25 cases of lymphoma and lung cancer found that 5% of patients experienced drug-related adverse events. However, 11 of 25 patients demonstrated stable or partial remission. This indicates the safety and efficacy of AZD9150. In the subsequent phase Ib clinical trial (NCT01563302), four out of 30 patients with therapeutic diffuse large B-cell lymphoma (DLBCL) achieved a four-month response, with a clinical ORR of 13% [193]. In 2016, the FDA approved BBI608, a small molecule that directly targets STAT3 DBD to inhibit STAT3 activation, for the treatment of tumors and pancreatic cancer at the gastroesophageal junction. In a phase III clinical trial of BBI608 for the treatment of advanced colorectal cancer (NCT0183062), the overall survival time of patients with high STAT3 expression was extended from three months in the placebo group to 5.1 months [194]. More small-molecule STAT3 inhibitors (YY201, TI-101, OPB-31121, and OPB-111077) and a PROTAC-degradation agent targeting STAT3 (KT-333) are currently in phase I/II clinical trials. In recent years, additional research has focused on developing small molecular inhibitors targeting STAT3, such as TI-101, OPB-31121, and OPB-111077, which have entered clinical trials (Fig. 5, Table 2).

Fig. 5

Schematic diagram of action mechanism of STAT3 direct inhibitor. Presently, the treatment strategy for STAT3 inhibitors in breast cancer mainly involves inhibiting the biological function of STAT3 protein directly or by inhibiting the mRNA level of STAT3 protein. A Direct inhibitors of STAT3 mainly target the SH2 or DBD binding domains of STAT3. These inhibitors can inhibit STAT3 transcription by inhibiting STAT3 dimerization or it's binding to DNA. B Some PROTAC drugs (SD-36) mainly degrade STAT3 protein, thus reducing it's expression levels [196]

Table 2 STAT3 inhibitors in clinical research stage

Currently, more inhibitors targeting STAT3 are being designed and developed. Small molecule STAT3 inhibitors and STAT3 degraders are the main areas of focus in research. However, combining STAT3 inhibitors with cancer treatment still has a long way to go. STAT3 inhibitors need to go through phase I, II, and III clinical trials to confirm their safety and efficacy before they can be used in clinical practice. It may still take 3–5 years before we see STAT3 inhibitors being applied in the field of cancer treatment.

Conclusions and perspectives

STAT3 is an important regulatory factor in the growth, differentiation, and survival of normal cells and their replication and division. Simultaneously, STAT3 is an essential regulator of growth-promoting receptor-positive breast cancer. Constitutive activation of STAT3 is closely related to the expression level of various breast cancer biomarkers. Moreover, STAT3 overexpression is associated with the poor prognosis (proliferation, invasion, and drug resistance) of patients, prompting an increasing number of breast cancer researchers to focus on STAT3. Blocking the biological function of STAT3 has also been proven to be a promising treatment for breast cancer [195].

STAT3 is also a transcriptional activator in growth-promoting receptor-positive breast cancer, regulating a variety of target oncogenes and influencing the progression, proliferation, apoptosis, metastasis, and drug resistance of breast cancer. In growth-promoting receptor-positive breast cancer, numerous upstream regulatory genes and downstream target genes of STAT3 have been identified, suggesting the potential for targeted therapies against STAT3 in the treatment of this subtype of breast cancer. Among these mechanisms, positive feedback loops and signal crosstalk are particularly notable, underscoring the role of STAT3 as a key node in the convergence of various signaling pathways. Targeting STAT3, either by indirectly inhibiting it through blocking upstream signaling pathways or directly suppressing it using peptides, oligonucleotides, and small molecules, primarily affects tumor development by interfering with STAT3 phosphorylation and preventing the formation of functional STAT3 dimers.

Early therapeutic trials have already validated the clinical relevance of targeting STAT3. Several small-molecule STAT3 inhibitors have entered clinical trials, some of which have shown promising results. In these trials, STAT3 inhibitors have been used either as monotherapies or in combination with chemotherapy or targeted therapies. To date, many STAT3 inhibitors have demonstrated significant efficacy in both in vitro and in vivo experiments, with some advancing to clinical trials. However, most small-molecule inhibitors have exhibited suboptimal clinical efficacy due to poor water solubility and cell permeability. In future research, PROTAC (proteolysis-targeting chimeras) may represent a novel approach for developing more effective STAT3 inhibitors. PROTACs not only inhibit the function of target proteins but also block their expression, offering dual action that confers stronger potency and higher specificity. This approach could overcome the limited clinical efficacy and potential toxic side effects observed with conventional STAT3 inhibitors. Further investigation into the STAT3 protein and exploration of the effects of PROTACs in different cellular environments and tumor types is warranted.

In recent years, several novel and specific STAT3 inhibitors have been discovered, showing reduced cytotoxicity to normal tissues and improved stability. The next step in STAT3 inhibitor research will focus on structural optimization of these inhibitors. STAT3 inhibitors, either used alone or in combination with other clinical therapies, may offer more promising outcomes in overcoming or reversing resistance to targeted therapies, such as anti-HER2 and anti-estrogen receptor treatments. Additionally, for breast cancer patients resistant to doxorubicin or capecitabine, STAT3 inhibitors may provide a better therapeutic benefit compared to traditional chemotherapy drugs. Therefore, STAT3 remains a potent clinical target for the prevention and treatment of breast cancer and warrants continued investigation.