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Osteosarcoma is a relatively uncommon cancer, accounting for 5 of childhood cancers and 8.9 of cancer-related deaths in children. The overall incidence is 5 cases per million persons per year .Despite modern multidisciplinary treatments including chemotherapy and surgery, the 5-year survival rate for patients diagnosed with osteosarcoma remains 60%-70% . Hence, there is a real need to develop novel approaches for treating osteosarcoma . Recent progress in identifying tumor-associated pathways and specific mediators of osteosarcoma pathogenesis, progression, and prognosis has led to novel targeted treatment approaches. However, there is no comprehensive summary about these progress and new molecular targets. So in this review, we discuss new molecular targets identified in the recent translational studies. These targets play roles in diverse aspects of osteosarcoma biology and include altered chromosomes and genes; regulators of cell migration and invasion, proliferation and apoptosis, angiogenesis, osteoclast function, and drug sensitivity; growth factors and signaling pathways; transcription factors; and miRNAs.
Altered chromosomes and genes
Osteosarcoma presents with complex karyotypes and a highly unstable genome exhibiting both numerical and structural chromosomal instability . Specifically, 1p35, 1q23.1-1q21.1, 6p22.1-6p21.31, and 19p13.11-19p13.2 are amplified, while 5q12.3-5q13.2, 5q14.3-5q22.2, and 13q13.2-13p14.3 are deleted. These regions include 1162 genes with high level amplifications and 146 with deletions . Some aberrations are correlated with clinical prognosis or are involved in important signal pathways, suggesting they partly result in tumorigenesis or progression .
KEGG pathway enrichment analysis of osteosarcoma revealed several key signaling networks in which multiple genes were altered at the chromosomal level. These include the vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) signaling pathways, in which amplifications occur, and the wingless-type MMTV integration site family (Wnt), cellular adhesion molecules (CAMs), and Hedgehog signaling pathways, in which deletions occur . Further investigation of the VEGF pathway revealed that VEGFA gene amplification is a poor prognostic factor for tumor-free survival of osteosarcoma patients . When used in combination with chemotherapy, the anti-VEGF antibody bevacizumab significantly improved survival and response rates in patients with metastatic colorectal cancer. Thus, anti-vascular therapy may hold promise for patients with osteosarcoma . Similarly, rapamycin andÂ its analogs, which target the mTOR pathway, inhibited cell growth in both humanÂ osteosarcoma cell lines and murine tumorÂ models . A recent study identified syndecan-2 as a WntÂ targetÂ and provided evidence supporting the pathologic role of Wnt signaling pathway inÂ osteosarcoma . In contrast, Cai and colleagues showed that the Wnt pathway is inactivated in osteosarcoma and therefore plays a potential tumor suppressor role, as stimulation of the pathway inhibits proliferation or promotes differentiation .
Osteosarcoma pathogenesis and progression has also been linked to inactivation of other tumor suppressor genes. The frequency of retinoblastoma 1 (RB1) and tumor protein p53 (TP53) alterations in sporadic osteosarcoma ranges from 30% to 40%, and patients with tumors harboring alterations in either gene seem to have poorer prognosis than those with tumors lacking such alterations . Recently, loss of WW domain containing oxidoreductase (WWOX) expression has been found in many cancers and has been associated with aggressive phenotype and poor prognosis, suggesting that WWOX inactivation contributes to cancer development and that WWOX is a tumor suppressor. The WWOX gene is deleted in 30% of human osteosarcomas, and the WWOX protein is undetectable in 61.8%, which indicates that loss of WWOX, whether by deletion of the gene or loss of protein expression, is likely an early event in osteosarcoma pathogenesis .
Another mechanism of osteosarcoma pathogenesis is oncogene aberration. APEX nuclease 1(APEX1) gene (14q11.2-q12) is amplified in osteosarcomas. APEX1 protein expression independently predicts local recurrence and/or metastasis, and APEX1 knockdown significantly suppressed the expression of VEGF. Thus, APEX1 is a candidate prognostic marker and therapeutic target for osteosarcoma . Increased copy number and protein expression ofÂ RecQ protein-like 4(RECQL4) (located in 8q24.4) has been reported as a frequent event in sporadic osteosarcoma .Â Similarly, Myc amplification has been implicated in osteosarcoma pathogenesis and chemoresistance. In a conditional transgenic mouse model of osteosarcoma, Myc inactivation caused proliferative arrest and promoted differentiation . A recent study showed that alternate splicing of MDM2 (also named HDMX) was superior to p53 mutation as a prognostic biomarker inÂ osteosarcoma.Â Moreover, the MDM2-S to MDM2-FLÂ ratio positively correlated with low MDM2 protein expression, rapid metastatic progression, and poor overall survival .
Regulators of cell migration and invasion
Molecules involved in osteosarcoma cell migration and invasion may be good therapeutic targets. Wan and colleagues reported that integrin beta-4, which is highly expressed in osteosarcoma cell lines and tumor samples, promotesÂ osteosarcomaÂ metastasis and interacts with ezrin. Suppressing integrin beta-4 expression or function was sufficient to revert the observed highly metastatic phenotype in the MNNG/HOS osteosarcoma cell model . Notably, ezrin contains a binding site for P-glycoprotein at residues 149-242 of band 4.1. Targeting this site may counteract drug resistance in osteosarcoma patients . Cyr61, a newÂ targetÂ of statins, is expressed at higher levels in osteosarcomaÂ than in normal bone and is further increased in metastatic tissues. Silencing Cyr61 inÂ osteosarcomaÂ cell lines enhanced cell death and reduced cell migration and invasion .
EarlyÂ in vitroÂ studies show that the Notch pathway down-regulatesÂ osteoclastogenesis/osteoblastogenesis and plays critical roles inÂ osteosarcoma. Recently, Zhang and collegues reported that Deltex1 blocks osteosarcomaÂ invasiveness by binding the intracellular domain of Notch and ubiquitinating Notch receptor. This mechanism may have important clinical implications for targeting Notch signaling inÂ osteosarcoma . Curcumin, a target of Notch, induced G2/M arrest in osteosarcoma cells, suppressing their growth. This inhibitory effect was accompanied by down-regulation of Notch-1 and its targets, including the MMPs, thereby preventing cell invasion. Thus, using curcumin to down-modulate Notch-1 signaling may be a useful strategy for osteosarcoma treatment .
Regulators of cell proliferation and apoptosis
In osteosarcoma, expression of ALDOA and/or SULT1A3 was significantly higher in patients with shorter survival time, suggesting these proteins are negative survival markers ofÂ osteosarcoma . Thus, these two molecules are potential targets in osteosarcoma. In addition, BCL2-associated athanogene 3(BAG3) alters the interaction between heat shock protein 70 (HSP70) and inhibitor of kappa B kinase gamma (IKK-Î³), increasing availability of IKK-Î³ and protecting it from degradation in SAOS-2Â osteosarcoma cells. This, in turn, increases nuclear factor kappa B (NF-ÎºB) activity and promotes survival. Treatment of xenograft mouse model with BAG3 siRNA reduced tumor growth and increased animal survival. These results indicate that BAG3 is a potentialÂ targetÂ for anticancer therapy in osteosarcoma .
In the MG-63 osteosarcoma cell line, transfection of an antisense Myc fragment induced cell cycle arrest and enhanced apoptosis . Using a conditional transgenic mouse model, Arvanitis and colleagues showed that Myc inactivation caused proliferative arrest and promoted differentiation in osteosarcoma. The proapoptotic molecule B-cell lymphoma 2 interacting mediator (BIM) also plays a role in apoptosis, autophagy, and anoikis in osteosarcoma. However, BIM alone may be insufficient for significant tumor cell death, as it is more likely to act in concert with other BH3-only proteins or death pathways . Knockdown of polo-like 1 kinase (PLK1) caused mitotic cell cycle arrest and subsequent apoptosis in several osteosarcoma cell lines and suppressed the growth of osteosarcoma xenografts in immunodeficient mice. Therefore, PLK1 is a promising molecularÂ targetÂ in osteosarcoma . Recently, Liang and colleagues reported that Î²-elemene induces apoptosis in osteosarcomaÂ cells in a dose- and time-dependent manner by increasing HIF-1Î± expression through reactive oxygen species (ROS) and the PI3K/Akt/mTOR signaling pathway. siRNA-mediated knockdown of HIF-1Î± or co-treatment with the HIF-1Î± inhibitor YC-1 significantly enhanced the antitumor effects of Î²-elemene. Therefore, a combination of Î²-elemene with HIF-1Î± inhibitor might be a useful therapeutic option forÂ osteosarcoma . Minibrain-related kinase (Mirk or Dyrk1B) is also a potential therapeuticÂ targetÂ in osteosarcoma. The overall survival of patients with high tumor expression of Mirk was significantly lower than those with low or moderate tumor expression of Mirk. Furthermore, Mirk knockdown inhibited growth and induced apoptosis in osteosarcoma cells .
Bcl-xL and midkine (MK) are also candidate targets for osteosarcoma therapy. For osteosarcoma patients, high levels of Bcl-xL mRNA expression are associated with poorer survival compared with low levels. Bcl-xL down-regulation or up-regulation significantly reduced or increased proliferation, respectively, inÂ osteosarcomaÂ cells . Furthermore, Bcl-xL down-regulation significantly enhanced chemo- or radiosensitivity of osteosarcomaÂ cells in vitro, and this might be associated with elevated caspase-3 activity . MK is highly expressed in osteosarcoma cells. MK silencing significantly induced apoptosis, whereas treatment with recombinant MK increased cell proliferation. Anti-MK monoclonal antibody suppressed osteosarcomaÂ cell growth and lung metastasis, suggesting it has potential therapeutic value .
Regulators of angiogenesis
The anti-VEGF antibody bevacizumab significantly improved survival and response rates in colorectal cancer patients . VEGFA is abundantly expressed in 74.1% of osteosarcoma cases, and patients with VEGFA-positive osteosarcomas had significantly worse tumor-free survival rates than patients with VEGFA-negative osteosarcomas . Thus, anti-vascular therapy might be effective for osteosarcoma patients. Endosialin expression has also been detected in HOS sarcoma cell lines, suggesting it too may be a therapeutic target for sarcomas .
Interestingly, Ichikawa and colleagues reported that venous thrombi adjacent toÂ osteosarcomaÂ harbor tumor surrounded by fibrin-expressing coagulation cofactors, which is associated with poor clinical outcome. More aggressiveÂ osteosarcomaÂ cell lines had greater surface expression of procoagulant factors, generated more thrombin, and proliferated in response to thrombin compared with their less aggressive counterparts. Treatment with low molecular weight heparin(LMWH) reduced in vitroÂ proliferation and procoagulant activity and in vivo tumor growth. These findings suggest that elements of the coagulation cascade may play a role in and be pharmaceuticalÂ targetsÂ to disruptÂ osteosarcomaÂ growth.
Growth factors and signaling pathways
Recently, Hassan and colleagues investigated cell surface receptor expression patterns in standard and patient-derivedÂ osteosarcomaÂ cell lines . IGF-2R was consistently overexpressed on the cell surface across all testedÂ cell lines. Substantial expression of c-Met, HER-2, IGF-1R, VEGFR-3, IR, and PDGFR-Î² was also detected, suggesting that these receptors may contribute toÂ osteosarcomaÂ aggressiveness and biological heterogeneity and may serve as potential targets . Similarly, Wang and colleagues reported that siRNA-mediated IGF-1R down-regulation suppressed osteosarcoma cell growth rates in vitro and reduced tumorigenicity in vivo. More specifically, IGF-1R down-regulation caused G0/G1 arrest and induced apoptosis via caspase-3 activation . Another study showed that IGF-2, not IGF-1, is the predominant growth factor produced by osteosarcoma cells, and that three different receptors [IR-A, HR(A), and IGF-1R] cooperate to form an IGF-2-mediated constitutive autocrine loop, in addition to the IGF-1R/IGF-1 circuit. Thus, cotargeting IGF-1R and IR-A is more effective than targeting IGF-1R alone in inhibiting osteosarcoma growth .
Nevertheless, targeting growth factors in osteosarcoma requires further investigation. A phase II trial tested trastuzumab in combination with cytotoxic chemotherapy for metastaticÂ osteosarcomaÂ withÂ HER2 overexpression. The 30-month event-free and overall survival rates were 32% and 59%, respectively, showing no significant improvement over the rates in patients without HER2 expression who received chemotherapy alone (32% and 50%, respectively). Although trastuzumab can be safely delivered in combination with anthracycline-based chemotherapy and dexrazoxane, its therapeutic benefit remains uncertain .
WNT5a and receptor tyrosine kinase-like orphan receptor 2 (ROR2), which are highly expressed in osteosarcoma, are both correlated with Enneking surgical stage and tumor metastasis. ROR2 knockdown with siRNA significantly inhibited proliferation and migration in osteosarcoma cell lines . Similarly, the physiological interaction of WNT5b and ROR2 can also enhance cell migration. Taken together, the WNT5b/ROR2 and WNT5a/ROR2 signaling pathways are promising therapeuticÂ targets forÂ osteosarcoma . Notably, Wnt5a/ROR2 signaling induces expression of matrix metalloproteinase 13 (MMP-13), and this effect is abrogated by an inhibitor of the Src-family protein tyrosine kinases (SFKs) . Thus, in addition to WNT5/ROR2, MMP-13 and SFKs are also potential therapeutic targets for osteosarcoma.
Regulators of osteoclast function
Better understanding of osteoclast regulation has accelerated the development of new therapies . The osteoclast, the sole cell that resorbs bone, is central in pathologic conditions in which bone destruction is intricately involved. OsteosarcomaÂ cells are of osteoblastic lineage, which is characterized by cells secreting the osteoclast-inducing factor receptor activator of NF-ÎºB ligand (RANKL). Receptor activator of nuclear factor-ÎºB-Fc (RANK-Fc), osteoprotegerin, bisphosphonates, and Src inhibitor control various aspects of osteoclast function. Hence,Â osteosarcomaÂ is a better candidate for osteoclast-targeted therapy than other primary and metastatic bone tumors .
Like Myc, which is involved in tumor cell cycle arrest and apoptosis , HSP90 is also a relevantÂ targetÂ for therapeutic intervention in osteosarcoma. The HSP90 inhibitor STA-1474 suppressed proliferation, reduced viability, and induced apoptosis in osteosarcoma cell lines and xenografts by down-regulating p-Met, p-Akt, and p-STAT3 and activation of caspase-3 .
Rb gene mutations in osteosarcoma result in increased activity of the transcription factor E2F1. In humanÂ primary osteosarcomas and E2F1-inducibleÂ osteosarcomaÂ cell lines, high E2F1 levels exerted a growth-suppressing effect (including p73 induction) that relied on the integrity of the DNA damage response network. Furthermore, proteome analysis coupled with bioinformatics revealed novel E2F1-regulated genes and signaling networks that may be useful targets in osteosarcoma .
STAT3 is also highly expressed in osteosarcoma tissues and is associated with poor tumor differentiation, metastasis, and low 5-year overall and relapse-free survival rates. RNAi-mediated STAT3 inhibition reduced proliferation and enhanced apoptosis inÂ osteosarcomaÂ cells. These effects were associated with inhibition of anti-apoptotic genes, including Myc and cyclin D1 . Cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata, was recently reported to inhibit the expression ofÂ STAT3 targetÂ genes, including anti-apoptotic Bcl-xL and cell cycle regulators Myc and cyclin D1. Moreover, in nude mouse xenografts of SaOS2 osteosarcoma cells, CEP significantly reduced tumor volume and weight . Thus, STAT3 is a potentialÂ molecularÂ targetÂ for osteosarcomas.
Yes-associated protein (YAP), a downstream target of Hippo signaling, acts as a transcriptional co-activator in the nucleus by associating with TEA-domain family member (TEAD). TEAD has a DNA-binding domain and regulates the expression of genes such as connective tissue growth factor (CTGF), amphiregulin (AREG), and cyclin D1 to induce cell proliferation. YAP expression and nuclear localization strongly influence cell proliferation and tumor promotion in several cancers. Thus, inhibiting YAP nuclear translocation and blocking target gene transcription may prevent tumor progression . Several studies show that YAP localization can be modified with chemical agents. For example, dobutamine binds to Î²-adrenergic receptors (Î²2AR) and promotes YAP phosphorylation at Ser127, causing YAP translocation to the cytoplasm through a Hippo- and Akt-independent mechanism .
Regulators of drug sensitivity
Recent data show that expression of hydroxysteroid (17-beta) dehydrogenase 10( HSD17B10), interferon induced transmembrane protein 2 (IFITM2), interferon induced transmembrane protein 3 (IFITM3), and ribosomal protein L8 (RPL8) is correlated with response to chemotherapy . In osteosarcoma, HSD17B10 expression was up-regulated in poor responders, and its protein levels in pretreatment biopsy samples correlated with response to chemotherapy . These data suggest that HSD17B10 may be a therapeuticÂ target in osteosarcoma. Myc gene amplification has been implicated in chemotherapy resistance . In addition, BCL2-like 11 (BCL2L11, also named BIM) mediates the antitumor effects of several chemotherapeutic agents, while BIM suppression supports chemoresistance .
Up-regulation of autophagy also plays a role in drug resistance. Autophagy, a catabolic process critical to maintaining cellular homeostasis and responding to cytotoxic insult, can either promote or inhibit antitumor drug resistance, depending on both the nature and duration of treatment-induced metabolic stress and the tumor type. InÂ osteosarcomaÂ cells, high mobility group box 1 protein (HMGB1)-mediated autophagy is a significant contributor to drug resistance. HMGB1 binds to the autophagy protein Beclin 1, regulating Beclin 1-PI3KC3 complex formation and autophagic progression. Therefore, HMGB1 is a critical factor in the development of chemoresistance and a novel targetÂ for osteosarcomaÂ therapy .
Spina and colleagues recently reported that inorganic phosphate (Pi) enhanced doxorubicin-induced cytotoxicity in U2OS osteosarcoma cells by inducing apoptosis. Remarkably, cytotoxicity induced by Pi/doxorubicin combination was accompanied by increased expression of p53 and the p53 targets MDM2, p21, and Bax. These effects were significantly reduced by the p53 inhibitor pifithrine-alpha. Moreover, the doxorubicin-induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Targeting Pi might, therefore, be a rational strategy for osteosarcomaÂ therapy .
MicroRNAs (miRNAs) are highly conserved, non-coding RNAs that simultaneously regulate multiple targets. Recent genome-wide screening using miRNA expression profiles has identified specific miRNA expression patterns of osteosarcoma. The identified miRNAs and their targets are potential novel biomarkers or therapeutic targets for osteosarcoma . The miRNA signature includes high expression of miR-181a, miR-181b, and miR-181c and low expression of miR-16, miR-29b, and miR-142-5p. Notably, expression of miR-181b and miR-29b was restricted to distinct cell populations in the tumor tissue, and high expression of miR-27a and miR-181c in pretreatment biopsy samples characterized patients who developed metastatic disease. In addition, high expression of miR-451 and miR-15b in pretreatment samples correlated with subsequent positive response to chemotherapy. In vitro and in vivo functional validation inÂ osteosarcomaÂ cell lines confirmed the tumor suppressive role of miR-16 and the prometastatic role of miR-27a, and further tests showed that their predicted target genes were down-regulated in osteosarcoma cells. These findings established an miRNA signature for osteosarcoma pathogenesis and revealed pretreatment biomarkers of metastasis and responsiveness to therapy .
Liu and colleagues reported that miR-125b is frequently down-regulated inÂ osteosarcomaÂ samples and cell lines. miR-125b suppressed osteosarcoma cell proliferation and migration through STAT3 down-regulation, suggesting that ectopic restoration of miR-125b expression might be promising treat approach . Also, miR-143 down-regulation promotes cellular invasion, probably via MMP-13 up-regulation, linking it to osteosarcoma metastasis to the lung . Recent studies show that miR-15a and miR-16-1 suppressed cyclinD1 transcription via direct binding to the cyclinD1 3'-untranslated region. Up-regulation of these miRNAs in SOSP-9607 osteosarcoma cells induces apoptosis and cell cycle arrest. The data suggest that repression of CCND1 by miR-15a and miR-16-1 is a new strategy for osteosarcoma therapy .
miRNAs in osteosarcoma are also implicated in chemoresistance. Cai and colleagues investigated the role of miR-215 in osteosarcoma cell resistance to dihydrofolate reductase (DHFR) inhibitor methotrexate. miR-215 suppressed denticleless E3 ubiquitin protein ligase homolog (DTL) expression, inducing G2 arrest and enhancing resistance to methotrexate. Thus, miR-215 may be an important regulator of chemoresistance and a novel therapeutic candidate .
Our understanding of the molecular basis of osteosarcoma has advanced considerably over recent decades. The translational studies described here provide an evolving list of candidate molecular targets and evidence in support of targeted therapy for osteosarcoma. Some of the factors identified in these studies function in several pathways and when targeted, may induce multiple responses. Furthermore, some factors may not affect osteosarcoma cells directly but may disrupt the complex biology between osteosarcoma cells and osteoclasts or tumor cells and endothelial cells in angiogenesis. As our knowledge of osteosarcoma at the molecular level continues to grow, optimal targeted treatment of osteosarcoma can be expected to follow in the future.
1. Osteosarcoma pathogenesis is complex and may involve genetic aberrations of the VEGF, mTOR, and Wnt signaling pathways; inactivation of p53, Rb, and WWOX; and amplification of APEX1, Myc, RECQL4, RPL8, HDMX, and VEGFA.
2. Recent translational studies that have shed light on new molecular targets for osteosarcoma have investigated genetic aberrations, tumorigenic pathways, key transcription factors, growth factors, miRNAs, and factors involved in tumor cell migration and invasion, proliferation and apoptosis, angiogenesis, osteoclast function, and drug sensitivity.
3. Several miRNAs (such as miR-215) and their target genes are potential novel biomarkers or therapeutic targets for osteosarcoma.
This work was supported by funds from the University Cancer Foundation via the Sister Institution Network Fund (SINF) at the Tianjin Medical University Cancer Institute & Hospital (TMUCIH), Fudan University Shanghai Cancer Center (FUSCC), and University of Texas MD Anderson Cancer Center (UT MDACC). This work was also partly supported by Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT) in China (IRT1076).