IL-6/JAK/STAT3 Signaling in Breast Cancer Metastasis: Biology and Treatment

Breast cancer is the most commonly diagnosed cancer in women. Metastasis is the primary cause of mortality for breast cancer patients. Multiple mechanisms underlie breast cancer metastatic dissemination, including the interleukin-6 (IL-6)-mediated signaling pathway. IL-6 is a pleiotropic cytokine that plays an important role in multiple physiological processes including cell proliferation, immune surveillance, acute inflammation, metabolism, and bone remodeling. IL-6 binds to the IL-6 receptor (IL-6Rα) which subsequently binds to the glycoprotein 130 (gp130) receptor creating a signal transducing hexameric receptor complex. Janus kinases (JAKs) are recruited and activated; activated JAKs, in turn, phosphorylate signal transducer and activator of transcription 3 (STAT3) for activation, leading to gene regulation. Constitutively active IL-6/JAK/STAT3 signaling drives cancer cell proliferation and invasiveness while suppressing apoptosis, and STAT3 enhances IL-6 signaling to promote a vicious inflammatory loop. Aberrant expression of IL-6 occurs in multiple cancer types and is associated with poor clinical prognosis and metastasis. In breast cancer, the IL-6 pathway is frequently activated, which can promote breast cancer metastasis while simultaneously suppressing the anti-tumor immune response. Given these important roles in human cancers, multiple components of the IL-6 pathway are promising targets for cancer therapeutics and are currently being evaluated preclinically and clinically for breast cancer. This review covers the current biological understanding of the IL-6 signaling pathway and its impact on breast cancer metastasis, as well as, therapeutic interventions that target components of the IL-6 pathway including: IL-6, IL-6Rα, gp130 receptor, JAKs, and STAT3.


INTRODUCTION
Breast cancer affects 1 in 8 women in the United States and is the second leading cause of cancerrelated deaths in women behind lung cancer (1). Breast cancers are diverse and often identified by molecular subtype via immunohistochemical (IHC) expression of prognostic markers: estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2). The molecular subtypes of breast cancer range in both receptor expression and prognoses: luminal A (ER and/or PR+ /HER2-), luminal B (ER and/or PR+/HER+), HER2-enriched (ER-/PR-/HER2+), and triple negative (ER-/PR-/HER2-) (2). HER2-enriched breast cancer and triple-negative breast cancer (TNBC) are the most aggressive subtypes represented by a higher Ki67 staining, poorer patient survival, and the highest propensity to metastasize (3). Primary breast cancer patients have a 5-year survival rate of 99%; however, the development of metastases diminishes survival rates to 28% (1). 20-30% of breast cancer cases metastasize to distant organs, which accounts for 90% of breast cancer-related deaths (4). The most common sites of metastasis for breast cancer patients include the bone, lung, brain, and liver (5). The development of metastases is a complex process comprised of multiple steps including epithelialmesenchymal transition (EMT), local invasion, migration, intravasation, extravasation, mesenchymal-epithelial transition, and colonization to a distant organ. During many of these steps, breast cancer cells secrete small soluble proteins, such as cytokines, to promote cancer cells (autocrine effect) and prime microenvironmental cells (paracrine effect) (6). Cytokines are secreted, pleiotropic proteins (15-20 kDa) that mediate a myriad of immunological and inflammatory responses that are often hijacked in cancer. Many cytokines can exhibit either pro-or anti-cancer properties, including interleukin-6 (IL-6). Under homeostatic conditions, IL-6 plays fundamental roles in immune response, inflammation, hematopoiesis, and bone homeostasis; however, dysregulation of IL-6 promotes the pathogenesis of multiple inflammatory and immune-mediated diseases, as well as, cancer (7,8). The IL-6 signaling pathway is one of the most dysregulated pathways in cancer. For example, IL-6 is elevated in sera of ovarian, cervical, colorectal, esophageal, head-and-neck, pancreatic, prostate, liver, lung, gastric, and breast cancer patients (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In breast cancer, IL-6 expression correlates with poor patient survival, promotes growth and invasion, and mediates metastatic progression, which identifies the IL-6 signaling axis as a potential therapeutic target (25,26). Consequently, many IL-6-pathway-targeted therapies have been developed and evaluated for breast cancer. Herein, we summarize the biology of the IL-6 signaling pathway, its roles in breast cancer metastasis, and therapeutic advancements in targeting the IL-6/JAK/STAT3 signaling axis.

Classic Signaling
Classic IL-6 signaling is mediated strictly through membranebound receptors, IL-6Ra and gp130 (Figure 1, left) (38). IL-6 first binds to IL-6Ra on the cell surface which creates a high affinity for transmembrane gp130. Two trimeric receptor complexes (IL-6/IL-6Ra/gp130) homodimerize; IL-6 of one trimeric complex binds to the D1 domain of gp130 of the second trimeric complex, forming a signal transducing hexameric receptor complex (39). The IL-6/IL-6Ra/gp130 receptor complex activates mitogen activated protein kinase (MAPK), phosphatidylinositide-3-kinase (PI3K), Janus kinases (JAKs), and signal transducer and activator of transcription (STATs) signaling cascades. Formation of the IL-6/IL-6Ra/ gp130 hexameric complex recruits the JAK family of nonreceptor tyrosine kinases (JAK1, JAK2, and TYK2) to the membrane which associate with and phosphorylate the cytoplasmic tail of gp130 at five tyrosine residues (Y759, Y767, Y814, Y905, and Y915) (40). Phosphorylated gp130 serves as a docking site for STAT1 and STAT3 transcription factors that are subsequently phosphorylated by JAKs at Y701 and Y705, respectively (41,42). Notably, IL-6 activates STAT3 more potently when compared to STAT1 (43). Upon phosphorylation, STAT3 undergoes a conformational change, detaches from the receptor complex, and homodimerizes allowing for STAT3 translocation into the nucleus to promote transcriptional activation (44). STAT3 is negatively regulated by tyrosine phosphatases, disruption of JAKs and/or cytokine receptors by suppressors of cytokine signaling (SOCS), or direct protein inhibitors of activated STATs (PIAS) (45)(46)(47). Receptor availability can also become a limiting factor for IL-6 signaling since IL-6 must be complexed with IL-6Ra in order to bind to gp130 receptor for signal transduction (38). Interestingly, transmembrane gp130 expression is ubiquitously expressed on most cell types; however, expression of membrane-bound IL-6Ra is restricted, therefore, limiting classic signaling to a small subset of cells (48,49). Since IL-6 modulates pleiotropic effects beyond immune cells, it quickly became evident that IL-6 signals via alternative mechanisms outside of membrane-bound receptors, termed trans-signaling.

Cluster Signaling
More recently, a third mechanism of IL-6 signaling has been reported where IL-6 signals between two interacting cells termed, trans-presentation or "cluster signaling" (37). Originally discovered in 2017, Heink et al. discovered IL-6 binds membrane-bound IL-6Ra on one cell (transmitting cell) and is able to bind a gp130 receptor on another cell type (receiving cell) for signal transduction (Figure 1, right) (37). Co-culture experiments identified that gp130 receptors on T cells responded to IL-6/IL-6Ra complexes on the membrane of dendritic cells resulting in robust activation of STAT3. Functionally, IL-6 cluster signaling allowed dendritic cells to prime pathogenic T helper 17 cells. Since sgp130 is known to antagonize IL-6 trans-signaling, but not classic signaling, Heink et al. also investigated whether sgp130 is able to neutralize IL-6 cluster signaling. Although sgp130 did not show inhibitory effects in this model, Lamertz et al. reported sgp130 to neutralize IL-6 cluster signaling by directly binding to the IL-6/ IL-6Ra complex on a transmitting cell (65). Given these contradictory findings, the IL-6 trans-presentation mechanism in addition to its biological and pathogenic roles remains to be characterized and elucidated.

IL-6 SIGNALING IN BREAST CANCER
Breast cancer is the most commonly diagnosed cancer in women. Despite recent advancements in targeted therapeutics, remission and survival in metastatic breast cancer patients remains poor (1). Metastatic dissemination can be regulated by multiple mechanisms including uncontrolled inflammation in the breast tumor microenvironment through the secretion of chemokines, growth factors, and cytokines to mediate immune evasion and promote tumor progression (66,67). Importantly, the tumor microenvironment is comprised of a myriad of cell types, such as tumor-associated macrophages (TAMs), helper T cells, bone FIGURE 1 | Overview of IL-6/JAK/STAT3 Classic, Trans-signaling, and Trans-presentation. Classic Signaling (left) occurs when IL-6 binds membrane-bound IL-6Ra leading to the subsequent formation of a trimeric receptor complex with signal-transducing subunit, gp130. Two trimeric IL-6/IL-6Ra/gp130 complexes bind through the D1 domain of gp130 to form a hexameric receptor complex for intracellular signaling through the JAK/STAT3 pathway. JAKs are recruited to the membrane and phosphorylate the cytoplasmic tail of gp130 and STAT3. pSTAT3 homodimerizes and translocates into the nucleus for activation of transcription. Trans-signaling (middle) occurs when IL-6Ra presents in a soluble form through mRNA alternative splicing or proteolysis by ADAM10/17. IL-6 binds sIL-6Ra to form a hexameric receptor complex through membrane-bound gp130 for signal transduction. Sgp130 antagonizes IL-6 signaling through sequestration of IL-6/sIL-6Ra. Transpresentation (right), or "cluster signaling," occurs between two different cells. A gp130 receptor complex on a receiving cell responds to a IL-6/IL-6Ra complex on a transmitting cell to induce downstream STAT3 signaling. marrow-derived cells, adipocytes, fibroblasts, and cancer cells which secrete pro-inflammatory cytokines, such as IL-6. IL-6 can be secreted in an autocrine or paracrine manner by both immune and non-immune cells in the tumor microenvironment. The mechanisms by which IL-6 mediates crosstalk between the tumor microenvironment and tumor cells continues to be investigated to develop therapeutic targets in breast cancer. Specifically, IL-6 is of particular interest due to its increased levels in sera of breast cancer patients when compared to normal sera or tissue of healthy patients (68,69). Increased IL-6 levels can be a result of single nucleotide polymorphisms (SNPs) in the promoter region of IL-6 gene, and have been demonstrated to predict poor prognoses in breast cancer patients. IL-6 SNPs are significantly associated with ER-positivity and results in a worse disease-free survival (70). Specifically, the rs1800795 SNP in IL-6 is associated with an increased risk of breast cancer metastasis, irrespective of ER status (71). Furthermore, IL-6 is upregulated in sera of patients with advanced stages of breast cancer and in patients presenting with metastases (68,72). Notably, patients with metastases at two or more sites have increased IL-6 sera levels compared to patients presenting with one metastasis, and high levels of IL-6 correlates with significantly worse survival in metastatic breast cancer patients (72)(73)(74). In addition to IL-6, clinicopathological analyses have been conducted on IL-6's corresponding receptor, IL-6R. Labovsky et al. identified IL-6 to positively correlate with IL-6R in breast cancer specimens, and that IL-6/IL-6R are co-overexpressed in breast carcinomas when compared to normal mammary tissues (75). Consequently, higher serum levels of sIL-6R predicts a shorter relapse-free survival in ER+ breast cancer patients (76). To complement above findings, stromal expression of IL-6Ra in primary breast carcinomas has been reported to be significantly correlated with metastatic occurrence, and a worse disease-free and overall patient survival (77). Moreover, IL-6 has also been reported to correlate with therapeutic resistance in breast cancer patients highlighting the IL-6/JAK/STAT3 pathway as an important prognostic marker in breast cancer progression, chemoresistance, and metastatic formation (78).

IL-6/JAK/STAT3 Role in Breast Cancer EMT and CSCs
Whether IL-6 enhances or inhibits breast cancer cell proliferation in vitro remained controversial for several years. Early studies reported recombinant IL-6 to inhibit or have no significant effect on breast cancer cell proliferation (105)(106)(107). More recently, it has become widely accepted IL-6 mediates an oncogenic role in multiple cancers, including breast cancer, primarily through the activation of STAT3. Since IL-6Ra and gp130 are required for signal transduction, the previous contradictory results may have been attributed to a lack of receptor expression in tested breast cancer cell lines, or that IL-6's pleiotropic effects may depend on JAK/STAT3 pathway activation. For example, gp130 suppresses cell-cycle progression by upregulating G 1 cyclin/cyclin-dependent kinase (CDK) inhibitor, p21, independent of STAT3. In stark contrast, gp130-induced STAT3 signaling regulates cell cycle transition from G 1 to S phase by upregulating cyclins D2, D3, A, and cell division cycle 25 A (CDC25A) while simultaneously downregulating CDK inhibitors, p21 and p27, indicating contradictory roles that may be regulated by the balance of STAT3 (108). STAT3 is involved in proliferation and suppression of apoptosis of breast tumor cells through the upregulation of target genes cyclin D1, c-myc, Mcl-1, Bcl-2 and Bcl-xL (92,109,110). STAT3 also upregulates MMP2, MMP9, Twist, Snail, and vimentin expression to mediate an EMT phenotype (87,95,111,112). To complement these findings, STAT3 knockdown resulted in a decrease in CD44+ subpopulation, mammosphere formation, and protein expression of stemness genes Oct-4 and Sox-2 in breast cancer cells (113). Sullivan et al. also reported IL-6 induces the EMT phenotype in breast cancer cells, and found IL-6 overexpressing breast cancer xenografts exhibited decreased E-cadherin and increased vimentin protein expression (114). More recently, Cho et al. utilized a microfluidic chip, which mimics the breast cancer microenvironment and breast metastatic phenotypes in vitro, and found that IL-6-treated breast cancer cells successfully invaded into blood and lymph vessels mimicking breast cancer lymphatic metastasis in vitro (115). Others have reported IL-6 to promote breast cancer cell proliferation, migration and invasion, and CSCs in breast cancer (116)(117)(118). Notably, breast CSCs are known to play a significant role in tumor recurrence and therapeutic resistance (119). Interestingly, mammospheres derived from node-positive breast carcinomas express higher levels of IL-6 when compared to their respective non-malignant matched mammary tissues indicating IL-6 may play a role in CSC renewal (120). Many studies have further validated IL-6 as a key regulator of breast CSCs. For example, IL-6 enriches the breast CSC subpopulation where administration of IL-6 stimulates spheroid growth in MCF-7 cells (120). To complement these findings, IL-6 enriches CD44+ cells as well as an EMT phenotype in breast cancer in vitro (114,121). To determine if IL-6-mediated breast CSCs translates in vivo utilizing mouse metastasis models, Korkaya et al. demonstrated PTEN knockdown activates the IL-6 inflammatory loop which, in turn, promotes the breast CSC subpopulation in HER2+/trastuzumab-resistant cells, tumor growth, and secondary metastases in vivo (122).
In addition to breast CSCs, surrounding microenvironmental cells are known to communicate with and prime secondary organs for metastatic dissemination. Multiple mechanisms mediate IL-6-induced breast cancer progression, such as activation of autocrine/paracrine loops under inflammatory conditions and IL-6's impact on the surrounding tumor microenvironment. For example, the oncogene Multiple Copies in T-cell Malignancy-1 (MCT-1), a recently identified prognostic biomarker in aggressive breast cancers, stimulates M2 macrophages in the tumor microenvironment through stimulation of IL-6. IL-6 promotes M2 macrophage polarization while concurrently stimulating TNBC stemness and tumor progression (123). IL-6 also functions in a paracrine manner to promote an invasive phenotype in breast cancer. For example, adipose stromal cells secrete IL-6 to promote breast cancer migration and invasion in vitro using the TNBC cell line, MDA-MB-231 (124). Further findings confirmed adipocyte-secreted IL-6 induces EMT in luminal A and TNBC cells through the activation of STAT3 (125). Additionally, isolated fibroblasts from breast tissue and breast cancer metastases secrete significantly more IL-6, enhance breast cancer cell growth, and induce pSTAT3 when compared to normal skin fibroblasts supporting IL-6's role in priming the "soil" for organ-specific metastasis (126). Taken together, the IL-6/JAK/STAT3 pathway is a major regulator of breast cancer metastasis through promoting breast cancer cell proliferation, EMT, enriching the breast CSCs, and suppressing apoptosis.

IL-6/JAK/STAT3 in Metastatic Breast Cancer Mouse Models
Given that the IL-6/JAK/STAT3 signaling axis promotes metastatic phenotypes of breast cancer, researchers have investigated the role of IL-6/JAK/STAT3 in breast cancer metastasis in vivo to identify key drivers and therapeutic interventions. IL-6 has been demonstrated to prime distant sites for metastatic formation. For example, tumor secreted IL-6 has been recently reported to enhance metastatic potential through educating monocyte-dendritic progenitors to prime distant organs for breast cancer metastasis. Magidey-Klein et al. demonstrates IL-6 plays a functional role in mediating crosstalk between primary tumors and the bone marrow to promote monocyte-dendritic progenitors to give rise to immunosuppressive macrophages which, in turn, promotes metastasis in vivo (127). Whether IL-6 mediates organ-specific breast cancer metastasis remains to be conclusively elucidated. However, due to the prominent role of IL-6 in bone metabolism and homeostasis, it is accepted IL-6 is associated with bone metastases and osteoclastogenesis. Dysregulated IL-6 promotes a pro-tumorigenic role in the bone microenvironment allowing breast cancer cells to invade the bone. Consequently, breast tumor cells secrete IL-6 in a paracrine manner to activate osteolytic target genes, namely, PTHrP, RANKL, and DKK-1 (128). In addition to breast cancer bone metastases, recent functional studies have demonstrated IL-6 to promote lung metastases in breast cancer in vivo. Siersbaek et al. conducted an intraductal xenograft implantation of an ER+ breast cancer cell line overexpressing IL-6 and found a significant increase in pSTAT3 in the primary tumors, as well as, an increase in metastases in the lung in vivo (129). Moreover, another group investigated metastatic potential of IL-6 via an intravenous injection of a TNBC cell line overexpressing IL-6, and found tumors to significantly increase lung metastases (86). To complement these findings, Lin et al. reports CGI-99, or C14orf166, enhances IL-6 transcription resulting in hyperactivation of IL-6/STAT3 signaling to promote lung metastases (130). There are multiple downstream effectors under current mechanistic investigation for their role in mediating IL-6-induced metastatic phenotypes in breast cancer. For example, Nyati et al. identified a novel downstream long noncoding RNA, AU021063, which is induced by IL-6 to promote breast cancer metastasis in vivo (131). While multiple models are are currently under investigation, mechanistic mouse models expand beyond the scope of this review. The emerging role of IL-6/JAK/STAT3 in promoting the breast CSC subpopulation and breast cancer metastases in vivo underscores the therapeutic potential in exploiting the IL-6/JAK/STAT3 signaling axis in metastatic breast cancer.

IL-6/JAK/STAT3 in Therapeutic Resistance
Since IL-6/JAK/STAT3 signaling upregulates breast CSCs which are known to mediate metastasis and therapeutic resistance, it is to no surprise that IL-6 has been shown to play a role in chemoresistance. IL-6 secretion and expression is significantly elevated in therapeutically resistant breast cancer cells when compared to their respective parental lines. Furthermore, administration of recombinant IL-6 induced chemoresistance through upregulation of drug-resistant gene, mdr1, in breast cancer cells (132). Wang et al. identified STAT3 induces breast CSC renewal and chemoresistance through upregulation of fatty acid b-oxidation; administration of leptin resensitized breast tumors to chemotherapy in vivo (133). Furthermore, activation of the IL-6 inflammatory loop induces trastuzumab-resistance in HER2+ breast cancer cells indicating that IL-6's proinflammatory role mediates breast cancer therapeutic resistance (122). Given these findings, studies have aimed to utilize IL-6/JAK/STAT3 signaling inhibition in combination with current standard-of-care (SOC) treatment (Tables 1, 2). Administration of Bcl-2 antagonist, sabutoclax, concurrently suppresses IL-6/STAT3 signaling to resensitize chemoresistant breast cancer cells to chemotherapeutic agents (175). Using a resistant ER+ patient-derived xenograft (PDX) mouse model, Siersbaek et al. reported a significant reduction in tumor growth with treatment of STAT3 inhibitor, ruxolitinib, but not fulvestrant alone, a SOC treatment for ER+ breast cancer (129). Clinically, cytoplasmic staining of IL-6Ra is significantly correlated with tamoxifen resistance in ER+ breast cancer patients suggesting IL-6/JAK/STAT3 is an actionable therapeutic target to sensitize tumor cells to current SOC treatment (78). To summarize, the IL-6/JAK/STAT3 signaling pathway mediates breast cancer progression, metastasis, and therapeutic resistance thus justifying investigations into IL-6/ JAK/STAT3 as a targeted therapy for breast cancer patients.

CURRENT THERAPEUTIC APPLICATIONS OF IL-6 SIGNALING IN BREAST CANCER
Due to the heterogeneity of breast cancer, molecular classifications help determine which tumors may respond to targeted therapy. Each breast cancer subtype corresponds to a different prognosis and treatment regimen. Patients with luminal A and B breast cancer subtypes typically respond to targeted treatments such as tamoxifen, fulvestrant, or aromatase inhibitors; luminal patients have the most treatment options and better prognoses (196). HER2-enriched breast cancers initially respond to anti-HER2 antibodies such as Food and Drug Administration (FDA)approved traustuzumab, lapatinib, pertuzumab, adotrastuzumab emtansinse, and fam-trastuzumab (197)(198)(199)(200), while TNBC patients have limited to no treatment options. Since TNBC tumors lack expression of ER, PR, and HER2, TNBC patients lack sensitivity to endocrine and molecular targeted treatments. Current SOC for TNBC patients includes systemic neoadjuvant chemotherapy and surgical resection (201). HER2-enriched breast cancer and TNBC are considered to be the most aggressive subtypes and maintain a higher propensity to metastasize (202). Metastatic HER2-enriched patients commonly acquire resistance to HER2-targeted therapies within one year, emphasizing the importance in developing novel therapeutics to treat or sensitize metastatic HER2-enriched tumors to current SOC treatment (203). Current findings elucidating the role of IL-6 in breast cancer progression, metastasis, and anti-cancer immunity, suggest the IL-6/JAK/STAT3 signaling pathway is an actionable target with preclinical and clinical studies demonstrating therapeutic potential in both primary and metastatic breast cancer. Notably, inhibiting the IL-6/JAK/STAT3 signaling axis has been investigated through directly targeting either IL-6, IL-6Ra, gp130 receptor, JAKs, or STAT3.

IL-6 Inhibitors
The FDA has yet to approve IL-6/JAK/STAT3 pathway inhibitors for breast cancer. However, monoclonal antibodies (mAb) and small molecule inhibitors are under preclinical ( Table 1) and clinical ( Table 2) investigation. Siltuximab is a chimeric IL-6 mAb which received FDA-approval for the treatment of multicentric Castlemans disease in 2014 (204). Morancho and others examined the efficacy of siltuximab in several PDX models, and found only two of the six lines responded to siltuximab treatment. Contradictory to previous findings, they did not find a significant reduction in pSTAT3 in all PDX cultures after inhibiting IL-6, indicating that identification of IL-6-dependent tumors is important for anti-IL-6 therapies to be efficacious (134). For example, serum IL-6 may be used as a biomarker for IL-6mediated treatment. Casneuf et al. analyzed the IL-6 serum levels of ERa-positive breast cancer patients and found IL-6 sera levels to be significantly correlated with intratumoral pSTAT3 protein expression. Furthermore, pretreatment of siltuximab reduced tumor growth in an ERa-positive breast cancer xenograft mouse model. Casneuf et al. also investigated a combination treatment using siltuximab and fulvestrant, and found combination treatment to attenuate tumor growth suggesting that IL-6/JAK/ STAT3 combination therapy may sensitize tumors to SOC treatment (135). MEDI5116 is a novel anti-IL-6 mAb which neutralizes IL-6, is efficacious against HER2+ trastuzumabresistant tumors, suppresses NF-kB signaling, and lung metastases (136). NF-kB promotes an IL-6 feed-forward inflammatory loop, whereas interruption of IL-6/NF-kB signaling may counteract IL-6-induced breast cancer chemoresistance and requires further investigation (102).

IL-6Ra Inhibitors
Multiple anti-rheumatic agents targeting IL-6, IL-6Ra, and JAKs have gained FDA-approval and have transformed treatment outcomes for autoimmune and inflammatory diseases. In 2010, tocilizumab was the first approved in the United States for the treatment of rheumatoid arthritis (RA). Tocilizumab is a humanized anti-IL-6Ra mAb that competitively binds IL-6Ra and disrupts the IL-6/IL-6Ra complex in both classic and transsignaling. Tociluzumab has a favorable safety and toxicity profile, and is now used for the treatment of juvenile idiopathic arthritis, adult-onset still's disease, giant cell arthritis, chimeric antigen receptor T cell-induced cytokine release syndrome, and systemic  In vitro findings using MCF-7 cells.
reported TNBC cells to secrete IL-6 in order to communicate with lymphatic endothelial cells to produce chemokine (C-C motif) ligand 5 (CCL5) to upregulate breast cancer lymph node metastasis. Combination treatment of tocilizumab and maraviroc, a CCR5 inhibitor, significantly reduces migratory and invasive phenotypes in TNBC cells in vitro, and breast cancer metastases in a TNBC xenograft mouse model in vivo (209). Given these results, tocilizumab proceeded to multiple clinical investigations ( Table 2). Tocilizumab was recently investigated in combination with trastuzumab and pertuzumab in metastatic trastuzumab-resistant HER2+ breast cancer patients in a Phase I clinical trial. This clinical study was completed March 20, 2020 and results are still under review (NCT03135171). Interestingly, tocilizumab is also under clinical investigation for severe COVID-19 treatment in breast cancer versus non-cancer patients where SOC chemotherapy may exacerbate severity of COVID-19 infection (NCT04871854). Tocilizumab in combination with atezolizumab and nab-paclitaxel is also under clinical investigation for safety of immunotherapy-based combination treatment in metastatic or inoperable locally advanced TNBC (NCT03424005). Sarilumab, an additional FDA-approved anti-IL-6Ra mAb for RA, which blocks both membrane-bound and soluble IL-6Ra, is under current Phase I and II clinical investigation in combination with capecitabine in stage I-III TNBC and metastatic TNBC patients (NCT04333706).
While IL-6Ra mAbs, tocilizumab and sarilumab, have made recent headway in clinical studies for breast cancer, drug repurposing remains an attractive therapeutic strategy to minimize the expensive, time-consuming drug development process (210). Diacerin, a non-steroidal anti-inflammatory drug used to treat osteoarthritis, directly interacts with IL-6Ra to suppress IL-6-induced phosphorylation of gp130, JAK1/2, STAT3, and MAPK in two TNBC cell lines. Furthermore, diacerin inhibits IL-6-induced STAT3 nuclear localization and transcriptional activity in TNBC cells, and significantly reduces tumor volume and induces apoptosis when compared to vehicle treated mice. Diacerin treatment reduces protein expression of IL-6Ra, pSTAT3, pMAPK, pAKT in TNBC tumor sections indicating diacerin could inhibit multiple IL-6-regulated oncogenic pathways (139).

gp130 Inhibitors
The gp130 receptor has evolved as an attractive therapeutic target to prevent downstream IL-6 signaling. Interestingly, small molecules which are FDA-approved for other therapeutic implications have been identified to have gp130 inhibitory effects. Since gp130 is the signal-transducing subunit for all IL-6 family cytokines, few gp130 inhibitors are able to maintain selectivity against IL-6. Bazedoxifene, an FDA-approved selective estrogen receptor modulator (SERM) with conjugated estrogens, was previously identified to reduce breast cancer cell proliferation and downregulate ERa and cyclin D1; however, its antitumor mechanism was not elucidated until recently (211). Interestingly, since the IL-6 family of cytokines bind different regions on the surface of gp130, bazedoxifene is able to selectively inhibit IL-6induced STAT3 in TNBC both in vitro and in vivo through direct binding of the gp130's D1 domain (144,145). Bazedoxifene was identified using a multiple-ligand simultaneous docking and drug repositioning approach in order to identify a small molecule that was able to directly bind into "hot-spot" residues on gp130 to prevent protein-protein interactions between IL-6 and gp130. Bazedoxifene inhibits STAT3-mediated transcriptional activity and, in turn, suppresses breast cancer colony formation, migration, and invasion. Bazedoxifene also reduces TNBC tumor volume suggesting the translational potential of the compound as an IL-6/JAK/STAT3 inhibitor (145). Whether bazedoxifene can inhibit IL-6-induced metastatic formation in TNBC is not known. Since bazedoxifene is FDA-approved with a favorable safety profile, bazedoxifene may provide clinical utility as a repurposed compound for the treatment of TNBC, but requires further investigation. Raloxifene, an additional FDA-approved SERM, has also been identified to directly bind to gp130 and suppress STAT3 activation in a TNBC cell line, SUM-159 (144). To further complement these findings, another group identified raloxifene to suppress breast cancer cell viability using another TNBC cell line, MDA-MB-231 (146). In 2007, raloxifene gained FDA-approval for the prevention of invasive breast cancer in postmenopausal women (212,213). Currently, raloxifene is not approved for treatment of breast cancer. Of note, bazedoxifene and raloxifene are both FDAapproved for the prevention of postmenopausal osteoporosis, and have been reported to prevent bone loss and increase bone mineral density (214). Since the bone is a common distant site of metastasis in breast cancer, and breast cancer bone metastatic patients suffer from microfractures and severe pain, raloxifene and bazedoxifene may provide additional benefits in addition to treating primary breast tumors (215).

STAT3 Inhibitors
STAT3 has gained significant attraction as an actionable anticancer therapeutic; however, there are currently no FDA-approved STAT3-targeted therapies for the treatment of cancer. Therefore, multiple studies have investigated novel small-molecule compounds which negatively regulate STAT3 activation in breast cancer (222) ( Table 1). Strategies for STAT3 inhibition include disruption of STAT3 phosphorylation, dimerization, nuclear translocation, or prevention of DNA binding. In 2006, stattic was identified as a small-molecule that disrupts the src homology-2 (SH2) domain of STAT3, and thereby prevents STAT3 recruitment to gp130 on the cell membrane (158). Functionally, increasing doses of stattic was able to prevent STAT3 dimerization and nuclear translocation resulting in a subsequent decrease in IL-6induced pSTAT3. Stattic induces apoptosis in TNBC cells (158). Since its discovery, others have identified stattic to be efficacious against breast CSCs through the downregulation of STAT3 stemness genes Oct-4, Sox-2, and Slug (113). Combination studies also reveal stattic is synergistic with SOC therapeutic, doxorubicin, and suppresses anti-apoptotic genes, Bcl-2 and Bcl-xL, to promote breast cancer cell apoptosis (159). Interestingly, another STAT3 inhibitor, STA-21, also directly binds to the SH2 domain of STAT3 to repress STAT3 transcriptional activity, and is efficacious in TNBC cells in vitro (160). Utilizing a structural-based computational screening approach, S3I-201 was identified to target the SH2 domain of STAT3 and suppress downstream signaling to induce breast cancer cell apoptosis and exhibit activity in vivo in a TNBC mouse model (172). Additionally, small molecule STAT3 inhibitors, FLLL31 and FLLL32, are derivatives of curcumin and selectively bind to the JAK2 and STAT3 SH2 domain. The JAK2 and STAT3 SH2 domain is essential for STAT3 phosphorylation, therefore, inhibition disrupts STAT3 dimerization and translocation required for activation of STAT3 transcriptional activity. Subsequently, downstream STAT3 target genes are significantly downregulated upon increasing doses of FLLL31 and FLLL32. FLLL31 exhibits efficacy in vivo where systemic administration reduced tumor growth and vascularity in a TNBC xenograft mouse model (161).
Other novel molecules have been identified to exhibit anti-STAT3 activity by inhibiting STAT3 phosphorylation. LLL12, prevents IL-6-induced STAT3 phosphorylation at Y705, and demonstrates efficacy in vivo in a TNBC MFP mouse model with a concomitant reduction in tumor volume and pSTAT3 expression (163). Interestingly, novel JAK2/STAT3 inhibitor, WP1066, can penetrate the blood-brain-barrier, suppress brain metastases in vivo, and prolong overall survival in mice inoculated with brain-trophic TNBC breast cancer cells via an intracardiac injection. WP1066 also reduces breast cancer cell viability and cell invasion in brain-trophic breast cancer cells through the reduction of STAT3 target genes, MMP-9 and VEGFR2 (94). Furthermore, Zinzalla and colleagues synthesized multiple pyrrolidinesulphonylaryl molecules and identified compound, 6a, to selectively inhibit IL-6-induced pSTAT3 in TNBC cells, and inhibit cell growth in STAT3dependent but not STAT3-null cells demonstrating its dependency on STAT3 for inhibition (162). Additional novel STAT3 inhibitors that have been investigated preclinically for the treatment of breast cancer include: LLY17, 6Br-6a, napabucasin, and coumarin-benzo[b]thiophene 1, 1-dioxide conjugates ( Table 1) (179,181,187,188).
Interestingly, multiple natural compounds have been identified to suppress STAT3 activity and exhibit anti-cancer properties. For example, CDDO-Me is a triterpenoid with antiinflammatory properties and suppresses activated STAT3 protein expression, nuclear translocation, and STAT3 antiapoptotic genes in ovarian and breast cancer in vitro (164). Other natural compounds which have exhibited anti-cancer properties in breast cancer through modulation of STAT3 activity include: naringenin, ilamycin C, esculentoside A, catechol, dihydrotanshinone, DT-13, cucurbitacin E, galiellalactone, schisandrin A, pectolinarigenin, eupalinolide J, betulinic acid, deguelin, picrasidine G, and cantharidin ( Table 1) (166-171, 173, 177, 180, 183, 185, 186, 190-192). Compounds have also been repositioned for the treatment of breast cancer due to their anti-cancer activity through inhibition of STAT3, and are under preclinical investigation. For example, niclosamide is currently FDA-approved as an anti-parasitic drug, yet treatment exhibited inhibition of IL-6-induced STAT3 activation resulting in suppression of adipocyte-induced EMT in breast cancer cells (176). Additionally, nifuroxazide, an antibiotic, exhibits anti-STAT3 activity and suppresses breast cancer tumor growth and lung metastases (178). Additional repurposed compounds under preclinical investigation for inhibition of STAT3 activity include pyrimethamine, flubendazole, and carfilzomib ( Table 1) (182,184,189). While STAT3 inhibitors have been extensively investigated preclinically, only one compound is under current clinical investigation. TTI-101 is a novel small molecule STAT3 inhibitor, and is in a Phase I clinical trial examining pharmacokinetics and compound safety in advanced breast cancer patients as well as patients with unresectable solid tumors (NCT03195699).

CONCLUSIONS
Under normal conditions, IL-6 is an important regulator in acute phase immune responses and modulates both anti-and proinflammatory reactions. Breast cancer cells can hijack the IL-6/ JAK/STAT3 signaling to evade normal immune responses and further promote tumor growth by activating surrounding microenvironmental cells. Therefore, it remains pertinent to maintain a homeostatic balance of IL-6/JAK/STAT3 as dysregulation creates a vicious autocrine and paracrine inflammatory loop which promotes breast cancer metastasis and therapeutic resistance. Recent reports extensively elaborate on IL-6's pleiotropic effects and pro-metastatic role in breast cancer; however, current evidence on whether IL-6 promotes site-specific metastases requires further investigation. Due to recent evidence of IL-6 inducing the CSC subpopulation and mediating therapeutic resistance in breast cancer, preclinical investigations in metastatic breast cancer focus on targeting this pathway with either mAbs, novel small molecule compounds, or by repurposing current FDA-approved compounds. Multiple actionable therapeutic targets reside in the IL-6 pathway including inhibition of IL-6 directly, IL-6Ra, gp130 receptor, JAKs, or STAT3. While there remains a plethora of preclinical studies analyzing IL-6/JAK/STAT3 inhibitors on breast cancer growth, there remains an urgent gap analyzing compound efficacy against breast cancer metastases in vivo. Additionally, since IL-6 modulates multiple physiological processes and oncogenic pathways, elucidating effective biomarkers for breast cancer patients who could benefit from targeted IL-6/JAK/STAT3 inhibitors could aid in the development of therapeutics for metastatic breast cancer patients.