STAT3 and its targeting inhibitors in osteosarcoma

Abstract Signal transducer and activator of transcription 3 (STAT3) is one of seven STAT family members involved with the regulation of cellular growth, differentiation and survival. STAT proteins are conserved among eukaryotes and are important for biological functions of embryogenesis, immunity, haematopoiesis and cell migration. STAT3 is widely expressed and located in the cytoplasm in an inactive form. STAT3 is rapidly and transiently activated by tyrosine phosphorylation by a range of signalling pathways, including cytokines from the IL‐6 family and growth factors, such as EGF and PDGF. STAT3 activation and subsequent dimer formation initiates nuclear translocation of STAT3 for the regulation of target gene transcription. Four STAT3 isoforms have been identified, which have distinct biological functions. STAT3 is considered a proto‐oncogene and constitutive activation of STAT3 is implicated in the development of various cancers, including multiple myeloma, leukaemia and lymphomas. In this review, we focus on recent progress on STAT3 and osteosarcoma (OS). Notably, STAT3 is overexpressed and associated with the poor prognosis of OS. Constitutive activation of STAT3 in OS appears to upregulate the expression of target oncogenes, leading to OS cell transformation, proliferation, tumour formation, invasion, metastasis, immune evasion and drug resistance. Taken together, STAT3 is a target for cancer therapy, and STAT3 inhibitors represent potential therapeutic candidates for the treatment of OS.


Abstract
Signal transducer and activator of transcription 3 (STAT3) is one of seven STAT family members involved with the regulation of cellular growth, differentiation and survival.
STAT proteins are conserved among eukaryotes and are important for biological functions of embryogenesis, immunity, haematopoiesis and cell migration. STAT3 is widely expressed and located in the cytoplasm in an inactive form. STAT3 is rapidly and transiently activated by tyrosine phosphorylation by a range of signalling pathways, including cytokines from the IL-6 family and growth factors, such as EGF and PDGF. STAT3 activation and subsequent dimer formation initiates nuclear translocation of STAT3 for the regulation of target gene transcription. Four STAT3 isoforms have been identified, which have distinct biological functions. STAT3 is considered a proto-oncogene and constitutive activation of STAT3 is implicated in the development of various cancers, including multiple myeloma, leukaemia and lymphomas. In this review, we focus on recent progress on STAT3 and osteosarcoma (OS). Notably, STAT3 is overexpressed and associated with the poor prognosis of OS. Constitutive activation of STAT3 in OS appears to upregulate the expression of target oncogenes, leading to OS cell transformation, proliferation, tumour formation, invasion, metastasis, immune evasion and drug resistance. Taken together, STAT3 is a target for cancer therapy, and STAT3 inhibitors represent potential therapeutic candidates for the treatment of OS.

| INTRODUC TI ON
Signal transducers and activators of transcription (STAT) proteins are latent cytoplasmic transcription factors that are activated by cytokines and growth factors. 1 Activated STATs translocate to the nucleus where they bind to promoter DNA elements and regulate gene transcription. 2 Seven STAT family members have been discovered in human and mouse: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. 3 STATs are cell signalling transducers for vital biological functions of cell growth, differentiation and survival. 4 STATs are conserved among eukaryotes and are involved with a wide range of functions including embryogenesis, immunity, inflammation, haematopoiesis and cell migration. 4 STAT3 is widely expressed and is transiently activated in response to epidermal growth factor (EGF) and interleukin-6 (IL-6) by tyrosine phosphorylation. 5,6 STAT 3 plays a crucial role in mediating cell growth, differentiation and survival signals of the IL-6 cytokine family via the gp130 receptor subunit. 4,7 STAT3 gene disruption leads to embryonic lethality in the mouse, indicating the vital role of STAT3 for mammalian development. 8 STAT3 is constitutively activated during the onset and progression of a variety of cancers, including multiple myeloma, leukaemia, lymphomas and solid tumours. 9 STAT3 overexpression is implicated in the development, progression and poor prognosis of osteosarcoma (OS) and emerges as a potential therapeutic target for the treatment of OS. [10][11][12] OS is the most common form of primary bone malignancy and the eighth most common childhood cancer, affecting approximately 2.4% of all childhood cancers. 13 OS has a bimodal age distribution with peaks during adolescence (10-14 years) and for adults aged over 65 years. 13 During adulthood, OS may occur as a second malignancy related to Paget's disease. 13 OS is thought to be derived from osteogenic progenitor mesenchymal or committed osteoblast precursor cells. 13,14 The 5-year survival rate for the treatment of OS is estimated to be 60%-70%, and poor prognosis depends on factors including the rate of metastases and chemotherapeutic resistance. 13,15 Here, we review the structure and function of STAT3, the role of STAT3 in OS and STAT3 inhibitors for the treatment of OS.

| Structure of STAT3
The human STAT3 gene is located on chromosome 17 (17q21.2) and has 24 exons. 16 The STAT3 protein was originally described as acute-phase response factor (APRF) and consists of six domains: an amino-terminus, a coiled-coil domain, the DNA binding domain, a linker domain, the Src Homology 2 (SH2) domain and a carboxyterminal transactivation domain. 17,18 Four STAT3 isoforms (α, β, γ and δ) have been identified. 19 The STAT3γ (72kDa) and STAT3δ (64kDa) isoforms are produced by proteolytic processing, and they appear to play an important role in the regulation of granulocyte development. 19 The STAT3α and STAT3β isoforms are produced by alternative splicing of exon 23 and have distinct biological functions. 16,20,21 Both STAT3α and STAT3β contain a tyrosine phosphorylation activation site (Y705) and SH2 domain within the C-terminus 22 ( Figure 1A,B). STAT3 activation by phosphorylation of Y705 leads to the formation of homo-or heterodimers via the SH2 domain and nuclear translocation for the regulation of gene transcription. 5,23 STAT3α (92kDa) contains two important phosphorylation sites (Y705 and S727) within the C-terminus ( Figure 1A). 20 STAT3 activation of transcription is maximal with dual phosphorylation of Y705 and S727. 24 STAT3α transcriptional activation of target genes may involve the recruitment of co-factors, such as CREB-binding protein (CBP)/p300, via the C-terminal transactivation domain. 25 STAT3β (83 kDa) is produced by alternative splicing, which results in frameshift coding for a truncated C-terminus lacking 55 amino acids and S727, which are replaced by seven amino acids and a stop codon 26 ( Figure 1B). STAT3α and STAT3β functional differences appear to be due to the presence or absence of the acidic C-terminal tail of STAT3α. 22 STAT3α appears to have greater transcriptional activity than STAT3β. 22 STAT3β appears to have a greater potential for constitutive activity, to bind DNA with greater affinity and to form more stable dimers than STAT3α. 22,23 The STAT3α acidic C-terminal F I G U R E 1 STAT3 secondary structure. STAT3 protein structure includes N-terminal domain, coiled coil and DNA binding domains, a linker domain, SH2 domain involved with dimer formation and the C-terminus. The C-terminus of the (A) STAT3α isoform and (B) STAT3β isoform confers distinct functions of the two isoforms. STAT3, signal transducer and activator of transcription 3 tail is thought to destabilize the active dimeric form and DNA binding of STAT3α, resulting in rapid dephosphorylation. 22 The STAT3α acidic C-terminal tail represents a potential mechanistic target for STAT3 deactivation. STAT proteins have distinct functional domains: the 130aa N-terminal domain mediates cooperative binding to multiple DNA sites, DNA binding specificity is conferred by residues 400-500 residues of the SH2 domain participate in dimer formation and the C-terminus is involved with the activation of transcription. 24,[27][28][29][30] Dimerized STAT proteins are generally thought to bind to DNA target sites via a 9-bp consensus sequence, TTCCGGGAA. 29 STAT3 tertiary structure may be considered in three domains and has distinct functional elements (Figure 2A-D). 31,32 STAT3 alphahelix 2 of the coiled-coil domain sequence element, R214/215, was shown to be required for nuclear translocation and subsequent export of STAT3. 33,34 STAT3 R214/215 appears to be the importin alpha5 binding site for nuclear translocation STAT3. 35 DNA binding sequence element, R414/417, is also required for nuclear translocation by stabilizing the STAT3 dimer for importin binding. 33,35 The STAT3 N-terminal and SH2 domains are potential targets for cancer therapy. The STAT3 SH2 domain contains three subpockets which represent potential therapeutic targets: the Y705-binding pocket, an L706-subsite and a unique STAT3 hydrophobic side pocket. 36 The STAT3 N-terminal domain is vital for function and contains a 4-helix bundle that is a potential target for cancer therapy. 29,37 STAT3 helix 2 analogs were rationally designed and demonstrated the potential to induce apoptosis of breast cancer cells. 37 Further research of STAT3 structural properties will improve the potential for diseasespecific targeted therapeutic applications.

| Biological function of STAT3
STAT3 is widely expressed and plays a vital role during mammalian development. 6,8 Expression analysis performed by Genevisible® across over 500 human tissues indicates leucocyte and T-cell populations are highly expressive of STAT3 ( Figure 3A). 38 Recent research indicates STAT3 protein is expressed in CD4 + T cells, T helper Th17 cells, Th1 and Th2 cells and the STAT3α isoform might interact with proteins, such as Prohibitin 1, for modulation of pathological immune responses. 39 STAT3 was originally identified as APRF for its role in mediating the acute-phase response in liver. 17 STAT3 was found to be rapidly activated by IL-6 for the regulation of acute-phase gene transcription. 17  interferon-γ. 5 STAT3 activation by IL-6 may be prolonged by binding of the IL-6 and EGF receptors, which may lead to constitutive STAT3 activity associated with disease. 49 STAT3 is activated by non-receptor tyrosine kinases, such as Src and Abl, via signalling pathways which are required for normal mitogenesis and may lead to oncogenic transformation. 50 The pathway of STAT3 activation by EGF effected by JAKs may depend on upstream Src kinase signalling. 46 STAT3 may be activated as a downstream effector of heterotrimeric guanine nucleotide-binding proteins (G proteins) signalling. 51 STAT3, together with ERK, activation is required during the Toll-like receptor-induced IL-10 production by B cells. 52 Taken together, STAT3 is activated both as a specific target and a downstream effector by a complex network of cellular signalling pathways to perform a wide range of biological functions. The disease-specific role of STAT3 activated signalling in the immune response, inflammatory disease and cancer warrants further investigation. STAT3 appears to play an important role in mediating cellular differentiation via its isoforms. 19 The STAT3 isoforms (α, β, γ and δ) are selectively expressed and activated during the regulation of granulocyte differentiation in vitro. 19 The ratio of STAT3 isoforms increased towards STAT3β with granulocytic differentiation and maturation. 19 The STAT3α and STAT3β isoforms are produced by alternative splicing and differ structurally and functionally. 16 21 The STAT3α and STAT3β isoforms appear to indicate the level at which granulocyte colony-stimulating factor (G-CSF) signalling diverges from immature normal to leukaemic human myeloid cells in vitro. 53 The ratio of STAT3α and STAT3β isoforms may affect the G-CSF-induced differentiation of myeloid cells. 53 The cellular signalling pathways of STAT3 isoforms remain largely unknown. Further investigation of the role of STAT3 isoforms in mediating cellular genetic programming in response to disease-specific external stimuli is required.

| THE ROLE OF S TATIN C AN CER S AND OS
STATs are important mediators of cell signalling for a wide range of biological functions including cell growth, differentiation and survival events of immunity and inflammation. STATs may be activated by oncoproteins and contribute to the process of malignant transformation by promoting cell proliferation and preventing apoptosis. 9 Dysregulated activation of STATs is frequently observed in human cancers. STAT3 constitutive activation is evident during the onset and progression of various cancers, including multiple myeloma, leukaemia, lymphomas and solid tumours. 9 Expression analysis by Genevisible® across over 500 human cancers indicates that STAT3 is highly expressed in metastatic carcinomas such as lung, breast and adenocarcinoma ( Figure 3B). 38 STAT3 dysregulation is thought to be involved with tumour progression, angiogenesis and metastasis. STAT3 appears to play a crucial role in the development of OS and represents a biomarker, prognostic indicator and a potential molecular target for OS gene therapy. 10 Further research is needed to investigate STAT3 as a predictive biomarker in prognosis and potential clinical therapeutic applications for cancers.

| The oncogenic potential of STAT3 in OS
STAT3 is implicated in the onset and progression of human cancers including multiple myeloma, leukaemia, lymphomas and solid tumours. 9 STAT3 overexpression is observed and associated with poor prognosis of solid tumours, such as gastric cancer, lung cancer, gliomas, hepatic cancers, OS, prostate cancer and pancreatic cancer. 54 STAT3 overexpression is significantly associated with poor prognosis of solid tumours, including OS, for three-and five-year overall survival. 54 OS is the most common primary malignancy of bone and the eighth most common childhood cancer. 13 OS may occur during adulthood as a second malignancy of Paget's disease. 13 The overall five-year survival rate of OS is estimated to be 68%. 13 STAT3 activation is abnormal in human cancers, such as OS, and transformed cell lines indicating the potential role of STAT3 in oncogenesis. 50,55 STAT3 was demonstrated as a proto-oncogene by the potential to mediate cellular transformation in vitro. 56 STAT3 constitutive activation directly by oncoprotein, Src, was shown to be a crucial signalling pathway for cell transformation in vitro. 57,58 Interestingly, the STAT3β isoform appears to abrogate gene induction by Src and to block cell transformation in a pathway-specific manner. 58  in highly metastatic murine OS K7M2 cells. 62 The role of STAT3 in OS stem cells (OSS) is largely unknown. Putative OSS markers include aldehyde dehydrogenase, CD133 and CD271. [62][63][64][65] activation may promote chemoresistance to OS, therapy by inhibiting the effect of chemotherapeutic agents on OSS. 63,64 Together, these findings suggest that STAT3 is a both a proto-oncogene and molecular target of known oncogenes implicated in the development of OS. STAT3 is overexpressed in OS tissues and cell lines and is a good predictor of poor response to OS chemotherapy. Further research is needed to investigate STAT3 and its isoforms as potential targets for OS gene therapy.

| STAT3 signalling pathways in OS
STAT3 signalling is implicated in the development and progression of OS (Figure 4). 9 STAT3 activation by cytokines, such as IL-6 and LIF, has been shown to promote the growth and metastasis of OS in vitro and in vivo. 66,67 Increased IL-6/JAK/STAT3 signalling is implicated in many human cancers and is associated with poor clinical prognosis. 68 Src/STAT3 signalling is upregulated in OS tissues and cell lines. 69 STAT3 activation was shown to mediate OS metastasis downstream of ΔNp63 in vitro. 70 ΔNp63 is a splice variant of p63, which blocks the tumour suppressor activity of p53, p63 and p73. 70 ΔNp63 was shown to upregulate IL-6/STAT3 signalling and promote angiogenesis of OS cells in vitro. 70  expression by miRNAs may be a potential avenue to identify OS biomarkers and therapeutic targets. STAT3 expression in OS was attenuated by miRNAs, miR-199a-3p and miR-340-5p, in vitro. 78,79 miR-340-5p overexpression resulted in decreased tumour size and weight in nude mice. 79 Further research is needed to determine the miR-340-5p/STAT3 signalling potentially involved with decreased OS tumour growth in vivo. STAT3 OS target genes may include cyclin D1, Bcl-2, Bcl-xL, survivin and Mcl-1, which represent potential targets for OS gene therapy. 59,80 The STAT3 signalling network involved with the progression of OS is diverse and pleiotropic. Further research is needed to identify vital STAT3 OS biomarkers and to develop potential therapeutic targets for STAT3-directed OS gene therapy.

| THE PATHOG ENE S IS OF S TAT3 IN OS
Constitutively activated STAT3 is associated with tumorigenesis and progression of many cancers, such as haematopoietic tumours (eg, multiple myeloma and leukaemia) and solid tumours (eg, breast cancer, lung cancer and pancreatic cancer). 81,82 STAT3 is considered to be an oncogene and may regulate oncogenic events involved with cell-cycle progression, apoptosis, tumour angiogenesis, invasion, metastasis and evasion of the immune system of OS. 82 STAT3 signalling represents a pathogenic pathway for the growth of OS in the bone microenvironment.

| Apoptosis
Dysregulated STAT3 activation has been shown to confer resistance to apoptosis in human cancers, including OS. 10

| Autophagy
STAT3 signalling might affect the cellular process of autophagy, with subcellular localization patterns indicative of its regulatory activity. 98 Nuclear STAT3 regulates the transcription of autophagy-related target genes during autophagy, such as BCL2, BECN1, PIK3C3, CTSB, CTSL, PIK3R1, HIF1A and BNIP3. 98 Nuclear STAT3 may also regulate the expression of microRNAs, which target autophagy-related genes. 99 Cytoplasmic STAT3 was found to inhibit autophagy by decreasing the activity of eukaryotic translation initiation factor 2-α kinase 2 via its SH2 domain. 100 Cytoplasmic STAT3 may also inhibit autophagy by sequestering autophagy-related proteins, FOXO1 and FOXO3. 101 Mitochondrial STAT3 is thought to complement nuclear and cytoplasmic STAT3, by suppressing autophagy and protecting against autophagic mitochondria degradation. 98,102-104 Mitochondrial STAT3 appears to limit oxidative stress and mitophagy by regulation of the electron transport chain and decreasing the production of reactive oxygen species. 103 STAT3 pathways regulating autophagy may provide therapeutic targets for OS treatment, indicating the need for further research.

| Drug resistance
STAT3 activation is attributed to both the progression and chemotherapeutic resistance of cancers, including OS. 81 The OS tumour microenvironment is implicated in the development of drug resistance and has been investigated. 105 Chemoresistance of OS cells appears to be influenced by MSCs within the tumour microenvironment. 105 IL-6/STAT3 activation was shown to regulate MSCs induction of chemoresistance in OS in vitro and in vivo. 105

| THE ROLE OF S TAT3 IN THE OS TUMOUR MICROENVIRONMENT
STAT3 is constitutively activated in tumour cells and immune cells of the tumour microenvironment in a range of cancers, such as OS. 108,109 STAT3 is a convergent mediator of oncogenic signalling by numerous pathways, and constitutive activation of STAT3 inhibits the expression of anti-tumour immune mediators, leading to an impaired immune response. 108 STAT3 signalling is involved with crosstalk between tumour cells, immune cells and the microenvironment, promoting tumour-induced immunosuppression. 108 STAT3 activity interferes with inflammatory signals of the immune system leading to immune evasion. 108,110 Constitutive STAT3 activity appears to inhibit the anti-tumour response by immune cells, including dendritic cells (DCs), T cells, natural killer (NK) cells and neutrophils. 109,110 STAT3 signalling between tumour cells, immune cells and the tumour microenvironment is mediated by factors, including IL-6, IL-10 and VEGF. 108

| Inflammation
Inflammation is associated with the onset and progression of cancers, including OS. [111][112][113] The inflammatory response influences each stage of tumour development from initiation and promotion to malignancy, invasion and metastasis. 111 Inflammation affects the immune cells response to cancers, immune cell interactions with cancer cells of the tumour microenvironment, and the response to cancer therapy. 111 STAT3 signalling activated by inflammatory factors, such as IL-6, cyclooxygenase-2 (COX-2) and TGF-β, appears to be involved in the development of OS. 67,114,115 Inflammatory factors may be secreted by cells of the tumour microenvironment, such as MSCs and macrophages. 67,114 Inflammatory cytokine IL-6/STAT3 signalling from MSCs in the tumour microenvironment appears to promote the survival, proliferation, metastasis and drug resistance of OS. 67,105 Further, constitutive TGFβ/IL-6/STAT3 activation, tumour growth and lung metastasis in OS is perpetuated by paracrine activity from tumour extracellular vesicle-educated MSCs (TEMSCs) in mouse and human OS tissue samples. 115 Inflammatory COX-2/ STAT3 signalling is upregulated by tumour-associated macrophages (TAMs) and promotes OS cell migration, invasion, and EMT in mouse and OS patients. 114 Together, these findings indicate that STAT3mediated inflammation influences the OS tumour microenvironment at each stage of tumour development leading to the progression, metastasis and drug resistance of OS. Cells from the tumour microenvironment, such as TEMSCs and TAMs, are potential therapeutic targets for OS therapy involving STAT3 signalling pathways.

| S TAT3 INHIB ITOR S IN OS TRE ATMENT
OS is a primary malignant bone tumour exhibiting aggressive growth and metastasis, leading to high mortality. The five-year survival rate of metastatic OS is estimated to be 20%-30% and has not improved significantly in the previous decades. 119,120 Targeted therapy for primary OS, metastatic OS and MDR OS to improve the prognosis of OS treatment is an intensive focus of research. STAT3 is a prime therapeutic target for OS treatment, and STAT3 inhibition appears to be a promising therapy for OS. 89 59 CDDO-Me appears to be a promising drug for OS therapy and requires further development of its clinical potential. 59 Small molecule inhibitor, LY5, was designed to prevent STAT3 homodimer formation by blocking the SH2 domain phosphotyrosine-binding site. 142 LY5 was shown to mediate anticancer effects on OS cells in vitro. 142 Further research is necessary to determine the signalling pathways mediating the anti-cancer effects of LY5. Anti-psychotic drug, pimozide, appears to be a novel STAT3 inhibitor with potential for OS treatment and requires further investigation. 143 Together, these findings suggest that direct STAT3 inhibitors are potentially effective for the treatment of OS and require further research to develop their clinical application.

Indirect inhibitors of STAT3
Indirect inhibition of STAT3 may be achieved by blocking upstream regulators of STAT3-signalling, such as IL-6 and EGF. 125 Inhibition of JAK/STAT signalling is an established strategy of targeting STAT3 in cancer therapy. 144 Tyrphostin AG490 is a Jak tyrosine kinase inhibitor and upstream inhibitor of STAT3 with therapeutic potential for OS. 145,146 AG490 has been shown to suppress the growth and induce apoptosis of IL-6-dependent multiple myeloma cells by downregulating the STAT3 and MAPK signalling pathways. 145 AG490 may be effective for the treatment of OS when combined with drugs, such as pterostilbene, and requires further investigation for the potential treatment of OS. 97 INCB018424 and cepharanthine (CEP)-701 are selective JAK inhibitors, which have been shown to suppress IL-6/STAT3 signalling, and have potential for the treatment of cancers, such as OS. 125,144,147 Irisin was identified as a hormone produced during exercise and it appears to have potential for the treatment of OS. 148,149 Irisin treatment demonstrated the reversal of IL-6 induced EMT in OS cells and inhibited the proliferation, migration and invasion of OS cells in vitro. 148 Irisin suppressed the IL-6 activation of STAT3 via the STAT3/Snail signalling pathway. 148 BBI608, or napabucasin, is a small molecule identified by its ability to inhibit STAT3-regulated transcription and cancer Natural compounds present an alternative to chemotherapy using agents, such as cisplatin, doxorubicin and methotrexate, and may offer hope to sufferers of OS with poor prognosis. Natural compounds appear to target signalling pathways involved in OS, such as JAK/STAT, PI3K/AKT, Notch and Wnt. 153 Natural compounds, including curcumin, diallyl trisulfide, resveratrol, apigenin, cyclopamine and sulforaphane may be grouped by their chemical structures and have potential therapeutic application in the treatment of OS.
Here, we focus on natural compounds currently thought to inhibit STAT3-signalling in the context of OS therapy.

Polyphenolic
Resveratrol is a natural polyphenolic compound, which may have potential for the treatment of OS. 64 Resveratrol was shown to inhibit OS cell viability and tumour growth in vitro via downregulation of the JAK2/STAT pathway. 64 Pterostilbene, a natural analogue of resveratrol with higher bioavailability, appears to be a potent inhibitor of OS cell growth via disruption of JAK2/STAT signalling, and its anti-OS effects may be enhanced when used in combination with AG490. 97 Pterostilbene has the potential to induce apoptosis of OS cells and requires further investigation for OS therapy. 97 Curcumin was shown to inhibit the proliferation and migration of OS cells via JAK2/STAT signalling, and synthetic curcumin analogue, FLLL32, is a potential anti-OS drug. 128,154 Chlorogenic acid is a polyphenol compound and was shown to inhibit OS cell growth and induce apoptosis in vitro via the STAT3/Snail pathway. 155 Further research is required to advance the therapeutic potential of polyphenolic compounds for OS.

Flavonoids
Flavonoids, such as ginkgetin, appear to have potential for the treatment of OS. 156 Ginkgetin, a biflavone extracted from the leaves of ginkgo biloba, was found to inhibit the growth and activate apoptosis of OS cells in a concentration-dependent manner via decreased STAT3 expression and activation of caspase-3/9. 156 Alkaloids Alkaloids, such as CEP, coptisine, sinomenine and columbamine, appear to have therapeutic potential against OS via inhibition of STAT3signalling pathways, and require further investigation. [157][158][159][160] Sinomenine was shown to inhibit OS cell invasion and metastasis by downregulating CXCR4/STAT3 signalling, resulting in decreased expression of OS target genes, MMP-2 and -9, RANKL and VEGF. 159 Sinomenine was shown to reduce OS progression and metastasis in vivo and appears to be a promising therapeutic agent for OS treatment. 159 Terpenoid Terpenoids, such as toosendanin, cucurbitacin B and I, RDA, glaucocalyxin A and catalpol, appear to have anti-OS potential via modulation of STAT3 signalling. [161][162][163] Toosendanin appears to inhibit OS tumour progression by preventing STAT3 dimerization and blocking STAT3/EGFR signalling. 161 Cucurbitacin B and I are novel STAT3 inhibitors with potential for OS treatment and require further investigation. 164,165 RDA was shown to inhibit OS cell growth and promote OS cell apoptosis in vivo by disruption of IL-6/JAK2/STAT3 signalling. 106 RDA may present a potential treatment option for doxorubicin resistance in OS. 106 Together, terpenoids require further research for their potential use in OS therapy. and anti-apoptosis gene, surviving. 166 Therefore, 4-MD may be a potential anti-OS therapeutic quinone compound and requires further investigation.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
STAT3 is a cytoplasmic transcription factor of the STAT family and is activated by tyrosine phosphorylation by numerous signalling pathways. STAT3 is activated by receptor tyrosine kinases, including cytokines of the IL-6 family signalling by the gp130 subunit, and growth factors, such as EGF, and non-receptor tyrosine kinases, such as Src and Abl. STAT3 may also be activated as a downstream effector of G protein signalling. STAT3 is a convergent intracellular signalling mediator for a range of pathways. Activated STAT3 is translocated to the nucleus for the transcriptional regulation of target genes involved with vital biological processes, including embryogenesis, immunity, haematopoiesis and apoptosis. The activation of STAT3 is rapid, transient and tightly regulated under physiological conditions. Four STAT3 isoforms have been identified, which have distinct biological functions. The role of STAT3 isoforms in cancers, including OS, is largely unknown and requires further investigation. Constitutive activation of STAT3 appears to lead to the onset and progression of OS. STAT3 is considered a proto-oncogene, and evidence suggests that dysregulated expression of STAT3 plays an oncogenic role in OS by promoting processes including cellular transformation, tumour growth, invasion, metastasis, resistance to chemotherapy and immune evasion.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.