Current research progress in targeted anti‐angiogenesis therapy for osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumour with a peak in incidence during adolescence. Delayed patient presentation and diagnosis is common with approximately 15% of OS patients presenting with metastatic disease at initial diagnosis. With the introduction of neoadjuvant chemotherapy in the 1970s, disease prognosis improved from 17% to 60%‐70% 5‐year survival, but outcomes have not significantly improved since then. Novel and innovative therapeutic strategies are urgently needed as an adjunct to conventional treatment modalities to improve outcomes for OS patients. Angiogenesis is crucial for tumour growth, metastasis and invasion, and its prevention will ultimately inhibit tumour growth and metastasis. Dysregulation of angiogenesis in bone microenvironment involving osteoblasts and osteoclasts might contribute to OS development. This review summarizes existing knowledge regarding pre‐clinical and developmental research of targeted anti‐angiogenic therapy for OS with the aim of highlighting the limitations associated with this application. Targeted anti‐angiogenic therapies include monoclonal antibody to VEGF (bevacizumab), tyrosine kinase inhibitors (Sorafenib, Apatinib, Pazopanib and Regorafenib) and human recombinant endostatin (Endostar). However, considering the safety and efficacy of these targeted anti‐angiogenesis therapies in clinical trials cannot be guaranteed at this point, further research is needed to completely understand and characterize targeted anti‐angiogenesis therapy in OS.


| INTRODUC TI ON
Osteosarcoma (OS) is the most common primary malignant bone tumour. It is characterized by a high incidence of metastasis at the time of diagnosis most commonly with early haematogenous lung metastasis, a poor prognosis and a high incidence of recurrence. The use of neoadjuvant chemotherapy with multiple agents has improved the 5-year event-free-survival (EFS) of non-metastatic patients to over 70% while reducing the incidence of amputation by 10%-20%. 1,2 However, with currently available treatment regimens, the overall 10-year survival rate is still less than 20% in metastatic patients. 3 Despite being known as a highly aggressive cancer, treatment for OS has remained essentially unchanged for more than 30 years, underscoring a critical need for new treatment strategies.
Compared with traditional treatment methods, targeted therapy is a type of high selectivity and low toxicity cancer treatment that uses drugs or other substances to precisely identify and attack certain types of molecules that are involved in the growth and progression of cancer (eg proteins, nucleic acid fragments and gene products). 4 Therefore,targeted therapies are currently the focus of much anti-cancer drug research and have been successfully applied to the treatment of chronic myeloid leukaemia, bowel, lung, breast and renal cancers. [5][6][7][8] In 1971, Folkman et al 9 first described the concept that tumour growth and metastasis can be inhibited by blocking angiogenesis, establishing the basis of anti-angiogenesis therapy. Several studies have reported the effective use of anti-angiogenic agents such as bevacizumab in breast and cervical cancer and sorafenib in liver and thyroid cancer. 10,11 In this review, we will be discussing the molecular pathogenesis of OS, giving insight to the specific molecules which may be targeted for the effective treatment of the disease.

| TUMOUR ANG I OG ENE S IS IN OS
Angiogenesis is a complex and highly adaptive process that is crucial for tumour growth and metastasis. The biological processes involved in angiogenesis include endothelial cell proliferation, differentiation and migration, recruitment of smooth muscle cells and maturation of blood vessels, with the processes being strictly controlled by vascular regulatory factors. 12 Vascular regulators include both angiogenic activators such as vascular endothelial growth factors (VEGF) and platelet-derived growth factors (PDGF), and inhibitors such as endostatin and angiostatin. An imbalance between vascular regulators will result in either angiogenesis or vascular degeneration. 13 More than that, locally produced vascular regulatory factors in the bone microenvironment are critical to the regulation of bone homeostasis. Dysregulation of angiogenic and angiocrine activities could also lead to altered bone homeostasis, which may contribute to tumour development in bone microenvironment. [14][15][16] Further, a hypoxic tumour cell environment has been reported to encourage angiogenesis in tumours by stimulating the overproduction of hypoxia inducible factor-1 (HIF-1) and VEGF. 17 The activation of endothelial cells by angiogenic factors leads to the production of proteolytic enzymes which degrade the extracellular matrix. The degradation of the underlying basement membrane enables endothelial cells to proliferate and migrate to the surrounding tissue to form new vessels. 18 These new vessels provide cancer cells with oxygen and nutrition and play an important role in cancer cell survival and metastasis. Thus, antiangiogenic therapies, aimed to suppress these processes, may provide an interesting approach in OS therapeutics.
As the most studied marker of tumour neovascularization, VEGF and VEGFRs regulate both the development of blood vessels from precursor cells during embryogenesis (vasculogenesis), and the formation of blood vessels from pre-existing vessels (angiogenesis). It has also been shown to promote endothelium proliferation, inflammation and vascular permeability. 22 The overexpression of VEGF is reported to be associated with disordered tumour neovascularization, destruction of endothelial cells, pericytes and basement membranes and has been implicated to promote cancer metastasis through the remodelling of microvasculature. 23 VEGF as a therapeutic target has been validated in various types of human cancers.
Over-expression of VEGF and the resultant increase in angiogenesis have been reported in a number of human cancers including OS. 24,25 VEGF levels correlate not only with the extent of tumour angiogenesis, but are a predictive measure of future metastases, and clinical prognosis. 26 Currently, the mechanism by which the majority of anti-angiogenic drugs prevent tumour angiogenesis is by inhibiting the VEGF or VEGF/VEGFR signalling pathway, causing tumour cells to 'starve' by disrupting its blood supply. 27 In addition, antiangiogenic drugs may also play an anti-cancer role in the treatment of osteosarcoma by targeting other targets ( Figure 1). The utilization of such angiogenesis inhibitors has been reported to be effective in pre-clinical trials and clinical treatments for OS (Table 1). 28,29

| ANTI -ANG IOG ENIC TARG E TED DRUG S FOR OS
Current management of non-metastatic OS is multimodal consisting of aggressive chemotherapy combined with radical surgical resection. Various combinations of adjuvant chemotherapeutic agents have been used, though the majority of recent trials still use highdose methotrexate, doxorubicin, and cisplatin (MAP). 30 The 'sandwich' therapy has reported 5-year EFS of 60%-70% for patients with non-metastatic OS. 31 However, patients with metastatic tumours that respond poorly to chemotherapy, or those with recurrent disease, still present a major clinical challenge. The survival rate for these patients still does not exceed 20% post-therapy with estab-

| Monoclonal antibody
Monoclonal antibodies (mAb) are laboratory engineered proteins which serve as substitute antibodies that can restore, enhance or mimic the immune system's attack on specific target cancer cells.   which correlated to no apparent improvement in EFS or overall survival. 44 Authors also reported that approximately half of the study cohort experienced a reduction in wound healing which is a complex process that includes a vascularization phase similar to that supports tumour growth with VEGF and VEGFRs as key regulators.

Bevacizumab
Bevacizumab has been shown to normalize tumour vasculature both in the laboratory and in patients with other types of cancers and therefore improve access for chemotherapy. In combination with other chemotherapeutic agents, it has been shown to be efficacious in the treatment of several cancers clinically by increasing tumour response and prognosis. However, there is a need for further investigation to the use of bevcizumab alone or in combination with other agents, to demonstrate its efficacy in OS patients.

| Sorafenib
Sorafenib is the first oral multi-kinase inhibitor that targets VEGFR-1, VEGFR-2 and VEGFR-3 to inhibit angiogenesis, the RET gene also known as RET/PTC rearrangement, RAF (including BRAFV600E) and platelet-derived growth factor receptors to inhibit tumour progression. 47

| Apatinib
Apatinib is a small molecule tyrosine kinase inhibitor that highly and selectively targets VEGFR-2 and also inhibits the activities of VEGFR-1, Kit, c-SRC and RET, 59  There are also ongoing clinical trials and unreported completed trials looking at pazopanib as a viable therapeutic option for OS patients. 71 So far, these studies have shown that pazopanib exhibited favourable clinical benefit and a tolerable toxicity profile compared to other TKIs.
These reports indicate that pazopanib has strong anti-tumour ac-

| Regorafenib
Regorafenib is a small molecule multi-kinase inhibitor, which inhibits

| Endostatin
Endostatin, isolated from a culture medium of rat endothelioma

| Traditional Chinese herbal medicine
In recent years, the synergistic effects of traditional Chinese herbal medicine (CHM) combined with radio-and chemotherapy have been gaining increasing significance as its effectiveness as an anti-

| SUMMARY AND FUTURE PROS PEC TS
There is no established systemic treatment option for advanced or unresectable OS progressing after standard chemotherapy, and as such, the survival rate of patients with OS has not improved significantly in recent years. Angiogenesis is a key factor affecting tumour growth and metastasis, and theoretically, anti-angiogenic therapeutic agents present potentially novel therapies for various cancer types.
Recently, progress has been made in the development and application of targeted anti-angiogenic drugs, providing much-needed relief in the search of therapeutic alternatives for OS patients.

ACK N OWLED G EM ENTS
Dr Yun Liu and Shijie Liao were visiting scholars to Jiake Xu' laboratory at the University of Western Australia.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest.

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.