Aurora-B: a novel biomarker in the invasion and metastasis of osteosarcoma

ABSTRACT

Osteosarcoma (OS), a primary human malignant tumor that affects the bones, mostly arises in children and adolescents. Even though surgical resection followed by radiotherapy and chemotherapy has improved the survival rate up to 60%, the long-term positive effect for most patients with OS is not satisfactory. Hence, elucidating the specific mechanisms involved in the pathogenesis of OS is particularly important. Aurora-B, a serine/threonine kinase, plays a crucial role in centrosome regulation, spindle formation and chromosomal separation during mitosis. It has been found that Aurora-B overexpression is related to the occurrence and development of several malignant tumors, including OS. This article summarizes the role of Aurora-B in the invasion and metastasis of OS.

Plain language summary

Article highlights.

• Osteosarcoma (OS) seriously affects the health of children and adolescents. It is urgent to search for novel biomarkers of OS to develop effective treatment strategies.

• Aurora-B influences the progress of mitosis, and has been found to be abnormally expressed in OS.

Biological functions of Aurora-B

• Aurora-B plays an important role in mitosis, including chromatin condensation, bipolar orientation and segregation and cytoplasmic division.

The expression & role of Aurora-B in OS

• Aurora-B is highly expressed in OS, and associates with tumor metastasis and worse prognosis of OS patients.

• Several factors affect the expression of Aurora-B.

Related mechanisms

• Let-7 cluster influences the development of OS by targeting Aurora-B.

• The phosphorylation of NPM1^Ser125 induced by Aurora-B promotes the malignant phenotype of OS through ERK/NF-κβ signaling pathway.

• PI3K/Akt/NF-κB signaling pathway plays a key role in the occurrence of OS.

• Autophagy activated by AMKP/mTOR/ULK1 signaling pathway could prevent OS progression.

Aurora-B as a potential drug target

• Five inhibitors, including VX-680, ZM447439, AZD1152, HOI-07 and APIO-EE-7, show good promise in the treatment of OS.

Conclusion & future perspective

• Aurora-B is a promising therapeutic target for OS, but more experiments in vivo and vitro with larger sample sizes are still needed for further validation.

1. Background

Growing from osteogenic mesenchymal stem cells, osteosarcoma (OS) is one of the most serious bone tumors in children and adolescents. It typically develops in the distal femur, proximal tibia and proximal humerus, which are long bones in the extremities [1,2]. With the use of neoadjuvant chemotherapy, the five-year survival rate of patients with OS has increased from 55 to 80%. However, due to the high recurrence rate of OS and multiple drug resistance, the prognosis of patients with OS is not very optimistic [3,4]. Considering the high recurrence, multidrug-resistance and metastasis of OS, there is an urgent need to identify novel biomarkers and elucidate their specific mechanisms in the development of OS, thereby developing more effective and precise treatment strategies for OS.

Aurora kinase family includes three members in mammalian cells, namely Aurora-A, Aurora-B and Aurora-C. Aurora kinase family, which is involved in multiple processes of mitosis, plays a crucial role in maintaining gene stability and regulating cell cycle [5,6]. Aurora-B, an important member of Aurora kinase family, can catalyze substrate phosphorylation by transferring ATP phosphate groups, regulating cell signal transduction, differentiation, growth and other biological processes [7]. Recently, a growing body of studies have demonstrated the abnormal expression of Aurora-B in malignant tumors, including OS, and related specific mechanisms have been gradually clarified with the progress of research. Treatment of OS with its small molecule inhibitors has taken a new turn, particularly when used in conjunction with chemotherapy medications. In this review, we first summarized the biological functions of Aurora-B and its relationship with OS, followed by the progress of inhibitors targeting Aurora-B.

2. Biological functions of Aurora-B

Aurora-B, encoded by the gene located in the 17p13, is an important member of chromosomal passenger complex (CPC). Its correct localization depends on the function of three other CPC members, namely Borealin, Survivin and INCENP [8,9]. It has been reported that Aurora-B plays a role in regulating chromatin condensation, bipolar orientation and segregation and cytoplasmic division during mitosis [10]. Aurora-B phosphorylates variant GENP-A on Ser7 and histone H3 on Ser10 in early G2, which causes chromatin to condense [11]. Accurate separation of chromatids requires correct connection between centromeres and microtubules. Aurora-B can phosphorylate the MCAK of the centromeres to inhibit microtubule depolymerization during mitotic spindle assembly. If the microtubules and centromeres are correctly connected, Aurora-B can maintain the stable state of correct connection by phosphorylating MCAK. Conversely, this role of Aurora-B will lost if incorrect connection exists, which may lead to the microtubule-centromere depolymerization and prevent the aggregation of the mitotic process [12–16]. At the end of mitosis, Aurora-B regulates cytokinesis by coordinating its action with Rho, an important kinase regulating cytokinesis [11,17]. Inhibition of the function of Aurora-B can seriously affect the formation of the central spindle during cytokinesis, resulting in cells being unable to divide normally and become polyploid cells. Consequently, when Aurora-B expression is abnormal, it will result in the formation of abnormal two-nucleon cells at the chromosomal separation stage from the two daughter cells that initially divide normally, which may promote carcinogenesis [18–20].

3. The expression & role of Aurora-B in OS

Overexpression of Aurora-B has been shown to cause multiple abnormalities in the process of mitosis, which may lead to cancer progression. It has been noted that a large number of malignant tumors express high levels of Aurora-B, such as lung, breast and bladder cancers, indicating that there may be a direct correlation between Aurora-B and the progression of tumors [21–23]. A recent study used tissue array analysis to profile the expression of Aurora-B in different tissues, and the authors identified that Aurora-B was overexpressed in OS compared with normal bone tissues [24]. In another study, Luo et al. [25] illustrated that the positive expression rate of Aurora-B in OS was significantly higher than that in osteochondroma. And the cells in mitotic stage had darker staining of Aurora-B, which indicated that Aurora-B was more strongly expressed in cells with active proliferation.

According to recent research, upregulation of Aurora-B was linked to an increase in tumor cell metastasis whereas downregulation of Aurora-B could prevent certain tumor cells from invasion and migration [26,27]. Liu et al. [28] reported that the positive rate of Aurora-B was markedly higher in OS with distant metastasis (78.6%) than in OS without distant metastasis (45.7%, p <0.05). Nevertheless, He et al. [29] discovered that OS cell proliferation, migration and invasive capabilities were inhibited when Aurora-B was knocked down, which also caused cell death and cell-cycle arrest at the G2 phase.

In addition to promoting the malignant phenotype, Aurora-B may affect the prognosis of patients with OS. Through Kaplan-Meier analysis, Wu et al. [24] showed that in OS patients, higher Aurora-B expression were inversely linked with both worse overall survival (p <0.01) and worse metastasis-free survival (p <0.01). Attempts have been made in another study to profile this connection and has similar results [30]. In conclusion, Aurora-B expression in OS is higher compared with other normal tissues, with a functional role of affecting the occurrence and development of OS. These suggest that Aurora-B as an oncogene may be a novel target for the therapy of this tumor.

Actually, the chromosome region where Aurora-B is located is prone to translocation, deletion and amplification, meaning the natural instability of the gene [16,31]. Hence, its abnormal expression or mutation will directly affect the level of Aurora-B. According to previous published papers, Aurora-B is regulated by transcription factors at the mRNA level and the abnormal process will affect its expression [32,33]. For example, it has been shown that the transcription factor, FoxM1, can regulate the expression of Aurora-B. In the late G2 phase of mitosis, the level of Aurora-B will increase if FoxM1 binds to the promoter region of Aurora-B, which may affect the progression of mitosis [34]. In addition, the abnormal degradation of Aurora-B is also one reason for its overexpression [35]. However, further studies are still needed to gain a deeper insight into the specific mechanisms underlying the overexpression of Aurora-B, as well as its potential association with OS.

4. Related mechanisms of Aurora-B in the invasion & metastasis of OS

4.1. Correlation between Aurora-B & let-7 cluster

MicroRNAs (miRNAs) are known as a class of tiny, non-coding RNA molecules with the length of around 22 nucleotides. By attaching to the 3′-untranslated regions (3′-UTRs) of target genes, mature miRNAs play important roles in the transcriptional and post-transcriptional control of gene expression [36,37]. Growing research has shown that miRNAs are crucial in controlling the expression of oncogenes and involve in cells growth, death and metastasis [38,39]. One of the miRNAs in the Caenorhabditis elegans genome that has been studied the most is let-7 cluster, which has been found to be strongly associated with the development of cancer [40–42]. Several studies have suggested the significant role of let-7 cluster in the progression of OS by targeting Aurora-B, including let-7a/g/i.

According to recent findings, compared with normal tissues, OS tissues had considerably lower levels of let-7a/g/i. As opposed to the cells in the group under negative control, let-7a/g/i up-regulated U2-OS and HOS cells showed considerably lower expression of Aurora-B mRNA and proteins, suggesting that let-7a/g/i could downregulate Aurora-B expression in human OS cells. Bioinformatical prediction and luciferase reporter assays revealed that let-7a/g/i had predicted binding sites in the 3′-UTR of Aurora-B, which mediated the suppression of Aurora-B. Moreover, at least partially by targeting Aurora-B in vitro, let-7a/g/i could suppress the malignant phenotype of OS cells [10,43–46]. These results indicated the potential role of let-7 cluster as a new biomarker for the diagnosis and treatment of OS by targeting Aurora-B.

In addition, Yu et al. [44] provided promising evidence that let-7a acted as a tumor inhibitor to inhibit NF-κβ/MMP signaling pathway by targeting Aurora-B in OS. NF-κβ, a member of the Rel family proteins, is essential in controlling protein production that mediates cell cycle/proliferation, anti-apoptosis and cytokine secretion [47]. MMP-2 and MMP-9 are two members of the matrix MMP family, which can degrade extracellular matrix and promote the invasion and metastasis of OS cells [48]. The activation of NF-κβ promotes tumor cell progression by increasing MMP-2 and MMP-9 proteins [49]. In this study, it was found that let-7a decreased the expression of NF-κβ, MMP-2 and MMP-9 proteins, and inhibited the malignant phenotype of OS by negatively regulating Aurora-B. These results suggest that targeting let-7a and Aurora-B/NF-κβ may be a potential treatment approach for the therapy of OS. Another study also confirmed this idea [46]. Research, however, on the role of let-7 cluster in the invasion and metastasis of OS is still at the preliminary stage, and more studies are needed to clarify the specific molecular mechanisms whereby let-7 cluster is involved in the progress of OS.

4.2. Correlation between Aurora-B & NPM1

NPM1 is a widely expressed nucleocytoplasmic shuttling protein, which is involved in DNA replication, transcription, ribosome assembly and chromatin remodeling. Post-translational modifications such as phosphorylation, acetylation and ubiquitination can localize NPM1 to different subcellular regions and perform different functions [50–52]. In recent years, accumulating studies have suggested that NPM1 is closely related to tumor progression.

Using the KinasePhos database, Pi et al. [53] found that NPM1 had serine and threonine phosphorylation sites, indicating a possible phosphorylation between Aurora-B and NPM1 since Aurora-B is a serine/threonine kinase. Further experiments in vitro showed that the expression of phosphorylated NPM1^Ser125 protein was decreased while the expression of non-phosphorylated NPM1 protein was unchanged after downregulating Aurora-B, suggesting that Aurora-B was able to mediate the phosphorylation of NPM1. The malignant phenotype validation in vitro revealed that silencing Aurora-B could inhibit the invasion, proliferation and migration of OS and this effect could partially alleviated by overexpression of NPM1. It indicated that Aurora-B could enhance the malignant phenotype of OS by mediating NPM1 protein phosphorylation.

The results above were also confirmed in another study [54], and found that the phosphorylation of NPM1^Ser125 induced by Aurora-B could activate the ERK/NF-κβ signaling, promoting the proliferation and metastasis of OS cells. ERK/NF-κβ signaling acts as an important factor in the malignant progression of tumors [55]. ERK1/2 is a highly conserved extracellular-regulated kinase, and its phosphorylation can induce the activation of NF-κβ in tumor cells. Then, the transcription of MMP-2 and MMP-9 initiates, enhancing the invasion, proliferation and migration of tumor cells by degradation of extracellular matrix [56]. This finding suggests that targeting the Aurora-B/NPM1/ERK/NF-κβ axis may be an innovative therapeutic option for the treatment of OS. However, more experiments are still necessary to investigate specific molecular mechanisms of the malignant progression of OS mediated by NPM1.

4.3. Correlation between Aurora-B & PI3K/Akt/NF-κB signaling pathway

PI3K/Akt is essential in both cell-extracellular matrix and cell-cell adhesion. Adhesion-dependent signals will be interrupted when lacks correct adhesion, leading to adhesion-related apoptosis [57]. The PI3K/Akt signaling has been proved an important role in the proliferation, invasion and migration of various tumors by influencing its downstream effect factors [58,59]. The phosphorylation of Akt has been considered to be a crucial regulatory factor of NF-κB activation. With the progress of research, the role of PI3K/Akt/NF-κB signaling pathway has been confirmed and attracted increasing attention in the occurrence and progression of OS [60,61].

Using immunohistochemistry to detect the expression of Aurora-B and p-Akt proteins in OS tissues from 24 patients with pulmonary metastatic disease, Zhu et al. [57] found a positive correlation between Aurora-B and p-Akt proteins (R = 0.726, p = 0.02), indicating a possible relationship between Aurora-B expression and Akt phosphorylation in OS. Further research showed that the levels of PI3K, p-PI3K (Tyr199) and p-Akt (Ser473) protein were significantly lower in cells with Aurora-B inhibition compared with the negative control group. Additionally, knockdown of Aurora-B significantly reduced the Akt phosphorylation, which in turn inhibited the expression of NF-κB. It indicated that inhibition of Aurora-B could prevent OS cells invasion and migration in vitro through PI3K/Akt/NF-κB pathway.

In another study, Pi et al. [62] found by western blot analysis that overexpression of Aurora-B increased the expression of PTK2, PI3K, Akt and NF-κB, thus speculating that Aurora-B could potentially enhance the malignant phenotype of OS cells through the activation of the PTK2/PI3K/Akt/NF-κB pathway. PTK2 is a cytosolic non-receptor tyrosine kinase that is involved in the oncogenesis and development of tumor cells. It is a significant regulator of integrin-regulated signaling [63,64]. Ren et al. [65] demonstrated that the malignant phenotype of OS cells was significantly inhibited by siRNA-based knockdown of PTK2. However, further research is needed to verify whether Aurora-B promotes the malignant phenotype of OS cells through activating the PTK2/PI3K/Akt/NF-κB signaling pathway.

4.4. The role of autophagy in Aurora-B-induced OS

Autophagy, also known as type II programmed death, is an evolutionarily extremely conserved process of intracellular self-digestion [66]. Autophagy is a complex biological behavior and plays a dual role in the progress of tumors [67]. In the early stages of carcinogenesis, autophagy acts as a tumor suppressor by reducing oxidative stress, preventing chromosomal instability and causing autophagic cell death. Later on, autophagy involves as a tumor activator, maintaining the homeostasis of tumor cells through anti-anoikis and metabolism-promoting activities. This ultimately leads to the spread and growth of the tumors [67,68]. In recent years, research on the function of autophagy in the emergence and development of tumors has gained significant attraction.

A study by Wu et al. [24] found that Aurora-B expression was negatively correlated with the level of autophagy-related protein LC3 in OS tissues. Additionally, the RFP-GFP-LC3 fusion assay demonstrated that autophagosomes and autolysosomes were higher in Aurora-B silenced cells than those in the negative control group. These findings showed that inhibiting Aurora-B could enhance OS cell autophagy. Another study also confirmed this connection and found that autophagy induced by Aurora-B knockdown could promote apoptosis of cells in OS [7]. Chloroquine, an autophagy inhibitor, could reverse this effect by inhibiting Aurora-B knockdown-induced autophagy.

Numerous signaling pathways, including mTOR and AMPK, play improtant roles in regulating autophgy. Recent studies revealed that the activation of autophagy depended on the alteration of AMKP/mTOR/ULK1 signaling pathway. AMPK is an essential stress and energy metabolism sensor that adversely controls mTOR, activating autophagy initiation factor ULK1 and enhancing autophagic flux [69–71]. Wu et al. [7] found that through activating ULK1^Ser555 phosphorylation, silencing Aurora-B could cause autophagy, promoting apoptosis in OS 143B cells. But bioinformatics analysis proved that there was no direct docking molecular structures between Aurora-B and ULK1, indicating that unknown molecules may exist and involve in intermediate regulatory links. Further study revealed that in Aurora-B-knockdown cell lines 143B and HOS, there was an upregulation of AMPK phosphorylation and higher expression of ULK1, whereas the expression of mTOR downregulated [24]. These results indicated that, by inhibiting the mTOR/ULK1 signaling pathway, silencing Aurora-B could promote autophagy. In addition, 143B and HOS cell migration and invasion could be greatly inhibited by silencing Aurora-B, but this effect could be reversed by MHY1485 (a mTOR activator). This suggested that activating mTOR/ULK1 pathway could revert the impact of Aurora-B inhibition on OS cell invasion and migration. These results are similar to another study [30]. In conclusion, silencing of Aurora-B can induce autophagy of OS cells partly through Aurora-B/mTOR/ULK1 axis, and this signaling pathway may be an effective treatment target and an indicator of prognosis for OS (Figure 1).

Figure 1.

The role of Aurora-B in the invasion and metastasis of OS (By Figdraw). (A) Aurora-B mediates NPM1 phosphorylation, activating the ERK/NF-κβ signaling pathway. The activation of NF-κβ initiates MMP-2 and MMP-9 transcription, promoting the invasion and metastasis of OS. (B) Let-7a downregulates Aurora-B expression, inhibiting NF-κβ/MMP signaling pathway. The decrease of MMP-2 and MMP-9 proteins prevents the malignant progression of OS. (C) Inhibiting Aurora-B influences the PI3K/Akt signaling pathway, reducing the Akt phosphorylation. Low p-Akt inhibites the expression of NF-κB, which in turn prevents the progression of OS. PETN: pentaerythrityl tetranitrate (a compound dephosphorylating PIP3); PI3K: a kinase phosphorylating PIP2. (D) Silence of Aurora-B promotes AMPK phosphorylation and ULK1 expression, while inhibits mTOR expression. The alteration of AMKP/mTOR/ULK1 signaling pathway activates autophagy, preventing the development of OS. MHY1485: a mTOR activator.

5. Aurora-B as a potential drug target in OS

At present, the widely accepted chemotherapy drugs for OS include methotrexate, cisplatin, doxorubicin and ifosfamide, but they have large toxic effects on the hematopoietic system, digestive system, etc. In this regard, there is an urgent need to develop more effective drugs with less side effects for the treatment of OS [72]. Due to the role of Aurora-B in tumor cell mitosis, it is a promising target for tumor diagnosis and treatment. To date, a growing number of inhibitors targeting Aurora-B have been developed as potential therapeutic agents. Several of them have entered clinical trials, while many are in preclinical research or development stages.

VX-680 and ZM447439, the first generation inhibitors of Aurora kinases, can inhibit their activity by blocking the ATP-binding sites. Both of them have achieved clinical validation in preclinical and I/II phases, and presented efficacy in several tumors with limited collateral toxicity to normal human cells [72–76]. In the preclinical validation, Tavanti et al. [77] revealed that human OS cell lines were highly sensitive to VX-680 and ZM447439, both of which variably caused apoptosis and hyperploidy in most cell lines. And VX-680 in combination with conventional chemotherapeutic agents could improve the clinical efficacy. These suggested their worth for further preclinical and clinical validation. AZD1152, the second generation inhibitor of Aurora kinases, is converted to AZD1152-HQPA rapidly in plasma, inhibiting the activity of Aurora-B selectively [78,79]. Many studies have shown that it is effective against several human tumors, including hepatocellular carcinoma and breast cancer [80,81]. It has also been confirmed to effectively inhibit the proliferation of human U2-OS cells by inhibiting the expression of Aurora-B in vitro [72,73]. However, in phase I studies, AZD1152 showed dose-limiting toxicity for individuals with neutropenia. Furthermore, in clinical trials, limited efficacy of AZD1152 for tumors was discovered due to the use of different Aurora kinase inhibitors [82,83]. These may reduce the value of its clinical application. Nevertheless, further clinical evaluation is still needed to determine its clinical efficacy.

In recent years, more and more inhibitors have been developed to inhibit the malignant phenotype of OS by targeting Aurora-B. Zhao et al. [82] identified a small molecule inhibitor called HOI-07. This compound could inhibit Aurora-B activity in OS, reduce the growth of OS anchorage-independent cells and cause G2-M phase arrest, inducing apoptosis as a result. Importantly, it was observed in the patient-derived xenograft (PDX) mouse model that HOI-07 treatment significantly inhibited the growth of OS without any significant toxicity. Jin et al. [84] identified a novel compound called APIO-EE-7, which can interact with Aurora-B at the ATP-binding pocket, inducing OS cell apoptosis and limiting their proliferation and colony formation. Moreover, in OS metastasis mouse model, it was found that APIO-EE-7 could significantly reduce tumor incidence and volume. At present, these two inhibitors are still in the laboratory development stage. However, before entering clinical trials, comprehensive evaluation and preclinical validation are needed to analyze their toxic reactions and drug resistance.

In general, the evidence indicates that Aurora-B represents a new candidate therapeutic target for OS. In the future, more drugs targeting Aurora-B to treat OS need to be developed and demonstrated through further clinical trials.

6. Conclusion

Aurora-B, a key kinase in the process of mitosis, is highly expressed in OS compared with normal bone tissues and its abnormal expression is closely related to the malignant phenotype of OS, which influences the prognosis of patients with OS as a result. Due to the high malignancy, hidden onset and rapid progression of OS, it is difficult to diagnose and treat this tumor in the early stage. As a promising anti-tumor target, Aurora-B provides new ideas for the treatment of OS. At present, there are many studies to explore the specific molecular mechanisms of the malignant phenotype of OS mediated by Aurora-B, and inhibitors that prevent the progression of OS by targeting Aurora-B have also been discovered. However, several limitations are still exist. Most studies have only clarified the effect of Aurora-B on OS through experiments in vitro. However, plenty of studies indicate that the tumor microenvironment plays an essential role in the malignant progression of tumors [85,86]. But effect of this factor is unable to be taken into consideration in vitro experiments, which may bias the results. Although some scholars have researched the effect of silencing Aurora-B on the change of OS tumor body in nude mice, studies of the effect on the metastasis of tumor cells in vivo remain vacant [87]. Therefore, more experiments in vivo are needed to investigate the effect of Aurora-B on the development, invasion and metastasis of OS. In addition, the sample sizes of OS tissues were small in most studies, which may affect the accuracy of the results. Therefore, larger sample sizes are needed for further investigation to explore whether Aurora-B can be used as a new molecular target for the treatment of OS.

7. Future perspective

At present, the treatment of OS mainly focuses on surgery and chemotherapy. Exploration of selective targeted anti-tumor drugs has always been the direction and goal of efforts. As a novel biomarker, Aurora-B has attracted extensive attention. In the future, more experiments in vivo and in vitro are needed to further prove the role of Aurora-B in the invasion and metastasis of OS, while expanding the sample sizes and optimizing the experimental design schemes. Although nude mouse models can well simulate the microenvironment of OS growth and metastasis in vivo, they lack thymus and mature cellular immunity, which cannot completely simulate the real immune microenvironment in vivo. In this regard, using genetically engineered mouse models to simulate the microenvironment in vivo is feasible to better optimize the experiments. Moreover, more experiments are needed to explore the upstream regulatory mechanisms of Aurora-B, such as whether there are epigenetic regulatory mechanisms that influence the expression of Aurora-B in OS patients. In addition, developing more specific drugs targeting Aurora-B and reducing their toxic side effects is also important for achieving the purpose of precise and effective therapy for OS.

Funding Statement

This work was supported by the Natural Science Foundation of Jiangxi Province, P.R. China (No. 20232ACB206043); Science and Technology Project of Jiangxi Provincial Health Commission, P.R. China (No. 20201017); and “Double Thousand Plan” Talent Project of Jiangxi Province, P.R. China.

Author contributions

All authors contributed to the study conception and design. JL suggested the topic of the manuscript. SL conceived the outline of the manuscript by searching the literature, wrote the major parts of the manuscript and created the figures. YZ and JL reviewed and edited the manuscript. Data authentication is not applicable. All authors have read and approved the final manuscript.

Financial disclosure

This work was supported by the Natural Science Foundation of Jiangxi Province, P.R. China (No. 20232ACB206043); Science and Technology Project of Jiangxi Provincial Health Commission, P.R. China (No. 20201017); and “Double Thousand Plan” Talent Project of Jiangxi Province, P.R. China. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.