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Advances in differentiation therapy for osteosarcoma
DRUDIS-2536; No of Pages 8 Drug Discovery Today Volume 00, Number 00 September 2019
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feature Advances in differentiation therapy for osteosarcoma Yingqian Chen1, Ji Cao1, Ning Zhang2, Bo Yang1, Qiaojun He1, Xuejing Shao1, [email protected] and Meidan Ying1, [email protected]
Differentiation therapy: from leukemia to osteosarcoma The concept of differentiation therapy was first introduced during the 1970s, as a means of inducing tumour cells to undergo terminal differentiation. The irreversible changes in the tumour phenotype make it possible to cure the cancer. Currently, differentiation therapy is mainly applied to haematological tumours. One typical example is the combination of retinoic acid (RA) and arsenic for acute promyelocytic leukemia (APL), which has become the standard clinical therapy for this disease. However, the application of differentiation therapy for solid tumours is lagging because of the complexity of the differentiation pathway. It is increasingly believed that solid tumours could also be treated with differentiation therapy, given a better understanding of the details and regulation of the differentiation process. Progress has been
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made with the use of differentiation therapy for solid tumours, especially osteosarcoma, as we discuss here. Osteosarcoma is the most common malignant primary bone tumour in adolescents and children, and is usually accompanied by lung metastasis. Osteosarcoma originates systematically from mesenchymal stem cells (MSCs) or committed osteoblasts and can be divided into osteoblastic, chondroblastic, and fibroblastic subtypes according to the predominantly differentiated component. In addition, osteosarcoma has significant heterogeneity because of its histological heterogeneity and instability of its genetic makeup [1]. Current treatment involves multidisciplinary treatment with neoadjuvant chemotherapy followed by surgery. However, clinical treatment faces numerous difficulties, including the adverse effects of chemotherapies, chemoresistance, and recurrence [2,3]. To overcome
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Differentiation therapy involves the use of agents that can induce differentiation in cancer cells, with the irreversible loss of tumour phenotype. The application of differentiation therapy in osteosarcoma has made progress because of a better understanding of the potential links between differentiation defects and tumorigenesis. Here, we review recent studies on differentiation therapy for osteosarcoma, describing a variety of differentiation inducers. By highlighting these examples of drug-induced osteosarcoma cell differentiation, we can acquire unique insights into not only osteosarcoma treatment, but also novel approaches to transform differentiating drugs into more effective therapies for other solid tumours.
these challenges, more effective treatment options are urgently needed. Given that most osteosarcomas are poorly differentiated histopathologically, differentiation therapy is viewed as a challenge in osteosarcoma treatment research.
Osteogenic differentiation defects The heterogeneous nature of osteosarcoma manifests by varying degrees of mesenchymal differentiation [4]. Moreover, osteosarcoma cells are considered to share similarities with primitive osteoblasts because of their undifferentiated or poorly differentiated stage and their self-renewal and proliferation abilities [5]. In particular, the more malignant phenotypes of osteosarcoma are often associated with characteristics of early osteogenic progenitors [6], suggesting that osteosarcoma is a differentiation dysfunction associated with osteogenic differentiation, which
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might have a role in osteosarcoma development and progression.
morphogenetic proteins (BMPs) [10] S100A4 [11], and S100A6 [12], are also involved. Some of these factors have become therapeutic markers for delineating the effects of differentiation therapy, including CTGF, Osterix, Runx2, osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP), and type 1 collagen [7]. In addition, proteins have been identified as differentiation markers, such as Wnt inhibitory factor 1 (WIF1) [13] and AT-rich sequencebinding protein 2 (SATB2) [14]. These molecules are key to understanding the cause of osteosarcoma and opening the door to differentiation therapy.
Osteogenic differentiation MSCs are bone marrow stromal cells that have the ability to proliferate, self-renew, and differentiate into mature tissues via osteogenic, chondrogenic, adipogenic, and myogenic differentiation [1]. Bone formation mainly refers to the process by which MSCs differentiate into osteoblasts, and the study of osteogenic differentiation is the most extensive and, thus, is our focus here. Osteogenic differentiation is an important process involved in bone formation, growth, and development. It is a well-coordinated multistep process that is regulated by a complex molecular framework [7]. Several transcription factors are involved, including Osterix, Runx2, Sox2, and Sox9 [8]. Other factors include secreted growth factors and their receptors, such as connective tissue growth factor (CTGF) and insulin-like growth factor I (IGF I). IGF I signalling is involved in normal bone growth and differentiation, and the mutations have been identified in IGF signalling genes in 7% of patients [9]. Bone formation-related proteins, such as bone
Associations between osteogenic differentiation defects and osteosarcoma
Differentiation therapy for osteosarcoma
A balance between the differentiation and proliferation potential of stem cells has been proposed [7]. When cells undergo differentiation, they lose their ability to proliferate, and are less sensitive to growth factors [15]. The pathogenesis of osteosarcoma occurs when abnormal osteoblast differentiation is disrupted by mutations or other causes and the cells maintain different stages of primitive osteoblasts. Growth
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PD-1/PD-L1
IGF
Interleukin
ra
tio
n
PI3K/AKT/mTOR
Pr
ol
ife
VEGFR
Bo
MMP En ne vi Im TNF-α ro m nm un en e ... t
Ce
ll
PDGFR
By defining osteosarcoma as a disease of differentiation dysfunction, we not only understand the pathogenesis of osteosarcoma from a new perspective, but also provide ideas for new therapeutics for osteosarcoma differentiation. Recent studies have focussed on overcoming the differentiation defects associated with osteosarcoma for therapeutic purposes. Examples of differentiation therapies are described below and summarized in Table 1.
Immunomodulators
Tyrosine kinase inhibitors TGF-β
factors then stimulate the precursor cells to proliferate uncontrollably, resulting in cancer development. Furthermore, osteosarcoma bears incomplete osteoblast differentiation characteristics, resulting in varying degrees of differentiation delay. In this context, there are close relationships between the defects in osteogenic differentiation and the pathogenesis of osteosarcoma. Other signalling pathways, such as cell proliferation, the bone immune environment, and hormonal milieu, are also involved in the development of osteosarcoma (Fig. 1). Therefore, differentiation therapy aims to induce differentiation by restoring the process of osteogenic differentiation in osteosarcoma cells.
...
M mo ili n eu al
Growth Hormone
re
or
Di ffe
H
SOX9 SATB1
Estrogen ...
Runx2
nt
ia
tio
n
BMP
PTH
PTHrP
SATB1
WNT
PPATγ Osterix Notch ...
Hormone modulators
Differentiation inducers Drug Discovery Today
FIG. 1
The main signaling pathways involved in osteosarcoma development and the potential therapeutic strategies. For definitions, please see the main text.
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TABLE 1
Summary of potential differentiation inducers in human osteosarcoma. Nuclear receptor agonists RARa agonists (ATRA etc.)
PPARg agonists
Hormone receptor modulators Estrogen receptor agonists PTH/ PTHrP and PTHR1 antagonists
Natural medicines Hyperoside Galangin Coleusin factor Oridonin Bufalin Other inducers GLP2 AsA/ascorbate
Trabectedin Alpelisib (BYL719)
Possible mechanisms
Underlying therapeutic strategies
Refs
Activates RARa; crosstalk with other pathways (MAPKs, NF-kB, TGF-b, Notch, Smad); inhibits M2 polarization of TAMs
RARa-based synergistic strategies: proteasome inhibitor; MDM2 enzyme activity inhibitor HLI373; sumoylation activator. Other synergistic strategies: PPARg agonists; methotrexate Synthetic compounds (thiazolidinedione, troglitazone, ciglitazone, pioglitazone); natural compounds (genistein, oridonin)
[6,17,20–24]
Activates PPARg; bypasses BMP pathway defects
Activates BMP pathway; promotes ER expression Regulates osteogenic differentiation: regulates the oncoprotein c-fos; inhibits PTHR1 pathway
DNA methylation inhibitor (decitabine); natural compounds (ugonin K, quercetin) PTH/ PTHrP: knockout or inhibition of PTHR1; Ezh2 inhibitors
[32–35]
Activates BMP-2 and TGF-b pathways; induces G0/G1 cell cycle arrest Activates (TGF)-b1, Smad2/3 pathway Activates BMP-2 pathway Activates PPAR-g; inhibits Nrf2 pathway Targets miR-148a; regulates DNMT1 and p27
N/A
[40]
N/A N/A N/A N/A
[41,42] [44] [30] [45]
N/A
[46]
Combined with As2O3
[47,48]
Combined with PD-1inhibitors
[49]
Combined with conventional chemotherapy
[50]
Inhibits proliferation rate; inhibits NF-kB; decrease c-Myc, PKM2, CyclinD1 Increases RUNX2 and SPP1 at low concentrations; induces apoptosis at higher concentrations through p21 Favours recruitment of Runx2; reduces proliferation rate Decreases cell proliferation by blocking cell cycle; affects osteoblast and osteoclast differentiation
Nuclear receptor agonists The nuclear receptor superfamily is a group of ligand-activated transcription factors, including retinoic acid receptor a (RARa) and peroxisome proliferation-activated receptor g (PPARg). RARa and PPARg agonists can regulate the terminal differentiation of osteosarcoma.
RARa agonists
[5,6,27–29]
RARa has a pivotal role in many differentiation processes by activating the transcription of downstream differentiation-related genes when binding to RA. All-trans retinoic acid (ATRA) is a vitamin A derivative with an important role in many processes, such as cell differentiation and embryo development [16]. The rapid clearance of APL in response to ATRA was a successful result of differentiation therapy. ATRA promotes osteogenic differentiation of osteosarcoma cells both in vitro and in vivo [17–19]. These results suggest ATRA to be a viable target to develop osteosarcoma treatment. RARa has a crucial role in ATRA-induced differentiation of osteosarcoma cells, and
researchers found phosphorylation of serine 77 and SUMO-1 modification of lysine 399 of RARa to be important for ATRA-induced cell differentiation [17,20]. In addition, ATRA can also crosstalk with other signalling pathways, such as the mitogen-activated protein kinase (MAPK), nuclear factor (NF)-kB, transforming growth factor (TGF)-b, Notch, and Smad signalling pathways [21]. ATRA also inhibits osteosarcoma cell metastasis by inhibiting the M2 polarization of tumour-associated macrophages(TAMs) [22]. These results provide a comprehensive understanding of the specific mechanisms of ATRA in the treatment of osteosarcoma. Although ATRA showed significant effects in preclinical studies in inducing osteosarcoma differentiation, its effects in patients with osteosarcoma have not yet been assessed (Table 2). This might be because the sensitivity of osteosarcoma cells to ATRA and the specificity of treatment remain to be elucidated. Recent studies attempted to further increase the sensitivity of osteosarcoma cells to ATRA and
[37–39]
improve its effectiveness in patients with osteosarcoma. Previous studies found that RARs undergo ubiquitination-mediated degradation, which could affect the ATRA-mediated transcriptional regulation of downstream genes. Therefore, low expression of RARa might be a key factor affecting the sensitivity of the clinical differentiation of osteosarcoma. Thus, attempts have been made to improve the differentiation treatment effect of ATRA by regulating the ubiquitination-mediated degradation of RARa. The transcription factor E2F1 and E3 enzyme MDM2 affect the stability of RARa, attenuating the osteogenic differentiation of osteosarcomas induced by ATRA [23]. Mechanistic studies revealed that MDM2 specifically regulates the degradation of RARa protein and the MDM2 enzyme inhibitor HLI373 effectively promotes osteosarcoma differentiation [24]. In addition to ubiquitination modification, the sumoylation of RARa is also important for its protein stability and protein nuclear localisation. It is speculated that the activation of sumoylation modification
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Differentiation agents
Features PERSPECTIVE IUPAC name
Structure
Clinical trials and preclinical models
Refs
Clinical trial
PDX/PDC
Cell lines/ xenografted animals
Cells lines
3,7-dimethyl-9-(2,6,6-trimethyl-1cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid
N/A
PDC
U2OS 143B, K7M2
U2OS
[17,21–24]
Trabectedin
Spiro [6,16-(epithiopropanoxymethano)7,13-imino-12H-1,3-dioxolo [7,8]isoquino [3,2-b] [3]benzazocine-20,1'(2'H)isoquinolin]-19-one,5-(acetyloxy)3',4',6,6a,7,13,14,16-octahydro-6',8,14trihydroxy-7',9-dimethoxy-4,10,23trimethyl-,(1'R,6R,6aR,7R,13S,14S,16R)-
Osteosarcoma, phase 2 (NCT00005625)
PDC
N/A
Saos-2
[49]
Decitabine
5-Aza-2'-deoxycytidine
Sarcoma, phase 1 (NCT02959164, NCT01241162)
N/A
143B
143B
[33]
AsA/ascorbate
Piperazine, 1-((1-phenylcyclohexyl)acetyl)4-(2-phenylethyl)-, coMpd. with L-ascorbic acid
Sarcoma, phase 2 (NCT03468075)
N/A
N/A
MG-63
[47,48]
Alpelisib (BYL719)
(2S)-N1-[4-Methyl-5- [2-(2,2,2-trifluoro-1,1dimethylethyl)-4-pyridinyl]-2-thiazolyl]-1,2pyrrolidinedicarboxamide
N/A
N/A
HOS, MOS-J
HOS, MG-63
[50]
Troglitazone
5-(4-((6-hydroxy-2,5,7,8tetramethylchroman-2-yl)methoxy)benzyl) thiazolidine-2,4-dione
N/A
N/A
N/A
143B, MG63
[5,15]
5-(4-((1-methylcyclohexyl)methoxy)benzyl) thiazolidine-2,4-dione
N/A
N/A
N/A
143B, HOS
[5]
Approved drugs ATRA
Agents in development Ciglitazone
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Differentiation inducers
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Differentiation inducers: clinical trials and preclinical models in osteosarcoma.
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TABLE 2
IUPAC name
Structure
Clinical trials and preclinical models
Refs
Clinical trial
PDX/PDC
Cell lines/ xenografted animals
Cells lines
5-((3S,5R,8R,9S,10S,13R,14S,17R)-3,14dihydroxy-10,13-dimethylhexadecahydro1H-cyclopenta [a]phenanthren-17-yl)-2Hpyran-2-one
N/A
PDC
N/A
MG-63
[45]
Oridonin
(14 r)-7-alpha,20-epoxy-1-alpha,6beta,7,14-tetrahydroxykaur-16-en-15-one
N/A
N/A
HOS
MG-63 HOS, Saos-2, U2OS
[30]
Galangin
3,5,7-trihydroxy-2-phenyl-4h-benzopyran4-on
N/A
N/A
MG-63
U2OS, MG-63
[41,42]
Genistein
5,7-dihydroxy-3-(4-hydroxyphenyl)-4Hchromen-4-one
N/A
N/A
N/A
MG-63
[29]
Quercetin
4H-1-benzopyran-4-one, 2-(3,4dihydroxyphenyl)-3,5,7-trihydroxy-
N/A
N/A
N/A
Mouse BMSC
[35]
Hyperoside
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl) oxy)-4H-chromen-4-one
N/A
N/A
N/A
U2OS, MG-63
[40]
Coleusin factor GLP2
N/A N/A
N/A N/A
N/A N/A
MG-63 MG-63
U2OS, MG-63 MG-63
[44] [46]
N/A N/A
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Bufalin
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TABLE 2 (Continued )
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of RARa could regulate the therapeutic effects of tumour differentiation. Several therapeutic strategies can be proposed for tumour differentiation based on the synergy between RARa post-translational modification regulation and ATRA: the proteasome inhibitor bortezomib, the MDM2 enzyme activity inhibitor HLI373, and a sumoylation activator could be used as ATRA sensitisers. In addition, RARa/MDM2/E2F1 could be a molecular marker for the treatment of osteosarcoma via differentiation therapy. Most of these outcomes are indicated by preclinical models but not yet by clinical trials, although this might only be a matter of time. ATRA can also synergize with agents to promote differentiation treatment effects, such as PPARg agonists [6] and methotrexate [25]. The latter is an antineoplastic agent that is currently in clinical trials for osteosarcoma treatment. It might be that ATRA would be a treatment for patients with osteosarcoma treated with highdose methotrexate (HDMTX).
PPARg agonists
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PPARg belongs to a nuclear receptor superfamily of PPARs and has an important role in osteogenic and adipogenic differentiation, cell proliferation, and apoptosis [26,27]. Exogenous expression of PPARg2, RXRa, and RARa, individually or in combination, inhibits in vivo osteosarcoma growth [6]. Additionally, some PPARg agonists can inhibit the proliferation of osteosarcoma cells and promote their differentiation in vitro, including thiazolidine derivatives, such as troglitazone and ciglitazone [5], which are currently used as insulin-sensitizing agents for antidiabetic drugs. PPARg agonists can bypass the defects in the differentiation pathway regulated by BMPs in osteosarcoma cell lines [28]. Some natural compounds can also promote the expression of PPARg and have a therapeutic effect, such as genistein [29] and oridonin [30]. Thus, the use of PPARg agonists is a good potential synergistic strategy for ATAR treatment.
Hormone receptor modulators Estrogen receptor agonists Estrogen is an important steroid hormone that is mediated by estrogen receptors (ER). Estrogen can regulate osteogenic differentiation and bone growth, facilitating bone formation and maintaining bone mineral density [31]. It can also induce the expression of genes related to osteogenic differentiation, such as those encoding BMPs and ALP [32]. In addition, the expression of ERa in osteosarcoma is significantly decreased 6
compared with that in normal tissues. Recently, the DNA methylation inhibitor decitabine was found to inhibit osteosarcoma cell proliferation and promote osteogenic differentiation via inducing ERa expression [33]. In addition, the natural compounds, ugonin K [34] and uercetin [35], were reported to have a role in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by acting in an ERdependent manner. These studies demonstrate the role of estrogen in regulating normal bone development and show that ER modulators act as corresponding differentiation inducers.
Parathyroid hormone and PTHR1 antagonists Parathyroid hormone (PTH) is a peptide hormone secreted by the parathyroid gland. PTHrelated protein (PTHrP) is similar in amino acid sequence to PTH. Both can bind to, and activate, PTH receptor 1 (PTHR1) to regulate bone differentiation and development and calcium homeostasis [36]. Both PTH and PTHrP can promote the differentiation of osteosarcoma cells [37] However, preclinical toxicology studies showed that a high incidence of osteosarcoma occurred with long-term use and high doses, but not with therapeutic doses of PTH administered to rats [38]. The correlation between PTH and osteosarcoma is intriguing. Recent studies found that knockout of PTHR1 affected osteosarcoma growth and promoted differentiation, whereas overexpression of PTHR1 had the opposite effect. This process was mediated by polycomb repressive complex 2 (PRC2), a finding supported by using Ezh2 inhibitors [39]. These studies suggest that inhibition of PTHR1 signalling is a useful way to increase differentiation and that PTHR1 antagonists are a potential treatment for osteosarcoma; however, the role of PTH in osteosarcoma requires further research.
Other potential differentiation inducers for osteosarcoma Natural compounds Many studies found that Chinese compounds can promote differentiation in osteosarcoma cells. For example, the flavonoid hyperoside, the main pharmacologically active compound in Hypericum perforatum, stimulates osteoblastic differentiation by activating the BMP-2 and TGFb pathways and inducing G0/G1 cell cycle arrest [40]. Galangin, the main active ingredient of Alpinia officinarum, activates the TGF-b1/ Smad2/3 signalling pathway [41,42]. With both these drugs, the antitumour effects are promoted by activating TGF-b to promote tumour
differentiation. Given the complicated role of TGF-b, such as its role in tumour metastasis, it cannot be defined as a therapeutic target, and further research is required [43]. Other flavonoids, such as ugonin K [34], quercetin [35], and genistein [29], have been verified as potential differentiation inducers for osteosarcoma. Likewise, the effects of terpenoids, including coleusin factor and oridonin, have been reported recently. Coleusin factor can inhibit the growth of osteosarcoma cells both in vitro and in vivo by activating the BMP-2 pathway [44]. [30] Finally, bufalin was found to inhibit the stemness and cell proliferation of osteosarcoma stem cells by directly targeting miR-148a, thereby regulating DNA methyltransferase 1 and p27 [45].
Other types of inducers In addition to natural medicines, other types of agent have clear effects on the differentiation of osteosarcoma cells, including peptides, and drugs currently used for other purposes. For example, glucagon-like peptide 2 (GLP2), a proglucagon-derived peptide, inhibits the growth of osteosarcoma cells and promotes the osteogenic differentiation of osteosarcoma cells by inhibiting NF-kB and decreasing levels of cMyc, PKM2, and CyclinD1 [46]. In addition, ascorbic acid (AsA)/ascorbate induces differentiation by increasing Runx2 and SPP1 gene expression and, at higher concentrations, it induces apoptosis through p21 in poorly differentiated osteosarcoma cells [47]. Huang et al. revealed the synergistic mechanism of As2O3 and AsA in human osteosarcoma [48]. Similarly, trabectedin recruits Runx2 and induces the striking differentiation of osteosarcoma cells. Combining trabectedin with immune checkpoint inhibitors and the accompanying enhanced expression of the inhibitory checkpoint molecule PD-1 has been suggested [49]. BYL719 (alpelisib) is a new a-specific PI3K inhibitor with dual functions in osteoblast and osteoclast differentiation and has been suggested to be combined with conventional chemotherapy for the treatment of osteosarcoma [50]. Thus, although these compounds or drugs were developed for other indications, they have been found to be useful as differentiation inducers for osteosarcoma.
Moving forward with differentiation therapy
Therapeutic potential in osteosarcoma Differentiation inducers for osteosarcoma have made significant progress in preclinical studies and some drugs approved for other indications have been the focus of clinical trials, such as
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Differentiation therapy and tumour heterogeneity Tumour heterogeneity makes it difficult to treat tumours, particularly osteosarcoma. Numerous studies have shown that ATRA can induce tumour differentiation in various cancers, including osteosarcoma, leukaemia, and neuroblastoma, which are paediatric cancers with obvious differentiation defects. Therefore, differentiation therapy might also induce the differentiation of different subtypes of osteosarcoma. Earlier studies reported that ATRA can induce different types of osteosarcoma, including fibroblast, chondroblast osteoblastic, and undifferentiated types [52]. Therefore, dif-
ferences in the classification of osteosarcoma might not affect the efficacy of differentiation therapy. However, the heterogeneous expression of differentiation therapeutic targets can affect the sensitivity of differentiation therapy. For example, RARa expression can lead to differences in the sensitivity of osteosarcoma cells to ATRA [23]. These results suggest that we need to determine the appropriate treatment population based on the therapeutic targets of differentiation therapy, similar to the concept of biomarkers. Therefore, as long as there are effective differentiation inducers and suitable biomarkers present, differentiation therapy will be more able to exert its effects in heterogeneous osteosarcoma or other paediatric cancers characterized by cellular differentiation. In recent years, there has been some progress in this area of research. For example, the regulatory mechanisms of osteogenic differentiation have been further elaborated, with various proteins revealed to be directly or indirectly involved in osteogenic differentiation and associated with the development of osteosarcoma. For example, Uev1A was reported to promote osteogenic differentiation of osteosarcoma by regulating the ubiquitination of the osteosarcoma-promoting factor Smad1. Uev1A overexpression not only promoted osteogenic differentiation of osteosarcoma, but also increased the sensitivity of osteosarcoma to chemotherapy [53]. In addition, human glioma pathogenesis-related protein 1 (GLIPR1) [54] and Meteorin, glial cell differentiation regulator-like (METRNL) [55] have both been reported to regulate the differentiation of osteosarcoma. Even though differentiation therapy for osteosarcoma has unique advantages and great clinical potential, it is progressing slowly. The crux of the problem lies in that there are too few known differentiation inducers, with little research on targets, especially for osteosarcoma. Moreover, in the context of the heterogeneity of osteosarcoma, there is a lack of biomarkers to determine the appropriate sensitive population for differentiation therapy. Thus, there is a need to use targeted ideas to study differentiation therapy in the future.
Concluding remarks Here, we have provided theoretical and preclinical evidence for the use of differentiation therapy for osteosarcoma. The discovery of molecules that can induce osteosarcoma to undergo terminal differentiation in vitro has promoted the use of differentiation therapy against osteosarcoma, with significant improvements in the treatment of solid
tumours. Researchers still have a long way to go to discover more effective differentiation inducers and biomarkers, as well as the indepth exploration of the molecular mechanisms involved. If osteosarcoma-induced differentiation mechanisms and regulatory factors can be elucidated, more differentiation-inducing agents and differentiation-inducing protocols can be proposed, rendering differentiation therapy a powerful weapon in the treatment of osteosarcoma. Differentiation therapy will also be an effective strategy for other solid tumours.
Acknowledgements This work was supported by the State Key Program of the National Natural Science Foundation of China (No. 81830107 to Q. He) and a grant from the National Natural Science Foundation of China (No. 81803552 to X.S.). We thank Chenghao Pan for help with drawing the chemical structures used in the figures. References 1 Mutsaers, A.J. and Walkley, C.R. (2014) Cells of origin in osteosarcoma: mesenchymal stem cells or osteoblast committed cells? Bone 62, 56–63 2 Isakoff, M.S. et al. (2015) Osteosarcoma: current treatment and a collaborative pathway to success. J. Clin. Oncol. 33, 3029–3035 3 Kansara, M. et al. (2014) Translational biology of osteosarcoma. Nat. Rev. Cancer 14, 722 4 Adamopoulos, C. et al. (2016) Deciphering signaling networks in osteosarcoma pathobiology. Exp. Biol. Med. 241, 1296–1305 5 Haydon, R.C. et al. (2007) Osteosarcoma and osteoblastic differentiation: a new perspective on oncogenesis. Clin. Orthop. Relat. Res. 454, 237–246 6 He, B.C. et al. (2010) Synergistic antitumor effect of the activated PPARgamma and retinoid receptors on human osteosarcoma. Clin Cancer Res. 16, 2235–2245 7 Wagner, E.R. et al. (2011) Defective osteogenic differentiation in the development of osteosarcoma. Sarcoma 2011, 325238 8 Almalki, S.G. and Agrawal, D.K. (2016) Key transcription factors in the differentiation of mesenchymal stem cells. Differentiation 92, 41–51 9 Behjati, S. et al. (2017) Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma. Nat. Commun. 8, 15936 10 Bragdon, B. et al. (2011) Bone morphogenetic proteins: a critical review. Cell Signal. 23, 609–620 11 Chen, X. et al. (2013) The E-F hand calcium-binding protein S100A4 regulates the proliferation, survival and differentiation potential of human osteosarcoma cells. Cell. Physiol. Biochem. 32, 1083–1096 12 Li, Y. et al. (2015) The calcium-binding protein S100A6 accelerates human osteosarcoma growth by promoting cell proliferation and inhibiting osteogenic differentiation. Cell. Physiol. Biochem. 37, 2375–2392 13 Baker, E.K. et al. (2015) Wnt inhibitory factor 1 (WIF1) is a marker of osteoblastic differentiation stage and is not silenced by DNA methylation in osteosarcoma. Bone 73, 223–232
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trabectedin, decitabine and ascorbate (Table 2). Given this progress, other approved drugs with appropriate preclinical reactions, such as RA and alpelisib, should be progressed to clinical trials in patients with osteosarcoma as soon as possible. What is the way forward for differentiation therapy in the treatment of osteosarcoma? Osteosarcoma is a highly aggressive cancer for which treatment has remained essentially unchanged for >30 years, with numerous problems of drug resistance, recurrence, and adverse effects of chemotherapy, suggesting that new therapies would be well received. In this context, targeted therapy and immunotherapy have attracted much attention in clinical and preclinical studies, and differentiation therapy, with its unique features, is another important choice for osteosarcoma treatment for several reasons: (i) it tends to be less toxic compared with conventional cancer treatments. Most differentiation inducers are physiological substances in the body, such as ATRA and ascorbic acid, and do not have obvious adverse effects on the body; (ii) given its unique ability to induce tumour cell differentiation, especially of cancer stem cells, differentiation therapy could be used for synergistic application with other therapies including chemotherapy, targeted therapy, and immunotherapy, to improve complete remission and cure rates. For example, ATRA can potentiate the effect of chemotherapeutic drugs, such as cisplatin and methotrexate [25,51]; and (iii) differentiation therapy can also activate antitumour immune responses, such as by inhibiting the M2 polarization of TAMs [22], thereby promoting immunotherapy effects. Therefore, as a new approach to the treatment of osteosarcoma that has low toxicity, differentiation therapy can provide a synergistic strategy with existing treatment systems, and has the potential to improve the survival rate of patients.
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14 Davis, J.L. and Horvai, A.E. (2016) Special AT-rich sequence-binding protein 2 (SATB2) expression is sensitive but may not be specific for osteosarcoma as compared with other high-grade primary bone sarcomas. Histopathology 69, 84–90 15 Wagner, E.R. et al. (2010) Therapeutic implications of PPARgamma in human osteosarcoma. PPAR Res. 2010, 956427 16 Cunningham, T.J. and Duester, G. (2015) Mechanisms of retinoic acid signalling and its roles in organ and limb development. Nat. Rev. Mol. Cell Biol. 16, 110–123 17 Luo, P. et al. (2010) Retinoid-suppressed phosphorylation of RARa mediates the differentiation pathway of osteosarcoma cells. Oncogene 29, 2772 18 Hisada, K. et al. (2013) Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes. J. Bone Miner. Metab. 31, 53–63 19 Dingwall, M. et al. (2011) Retinoic acid-induced Smad3 expression is required for the induction of osteoblastogenesis of mesenchymal stem cells. Differentiation 82, 57–65 20 Zhou, Q. et al. (2014) Small ubiquitin-related modifier-1 modification regulates all-trans-retinoic acid-induced differentiation via stabilization of retinoic acid receptor alpha. FEBS J. 281, 3032–3047 21 Yang, Q.J. et al. (2012) All-trans retinoic acid inhibits tumor growth of human osteosarcoma by activating Smad signaling-induced osteogenic differentiation. Int. J. Oncol. 41, 153–160 22 Zhou, Q. et al. (2017) All-trans retinoic acid prevents osteosarcoma metastasis by inhibiting M2 polarization of tumor-associated macrophages. Cancer Immunol. Res. 5, 547–559 23 Zhang, L. et al. (2014) E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma via promoting ubiquitination-mediated degradation of RARalpha. Cell Cycle 13, 1277–1287 24 Ying, M. et al. (2016) The E3 ubiquitin protein ligase MDM2 dictates all-trans retinoic acid-induced osteoblastic differentiation of osteosarcoma cells by modulating the degradation of RARa. Oncogene 35, 4358 25 Sramek, M. et al. (2016) Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell differentiation. Cancer Cell Int. 16, 14 26 Zhuang, H. et al. (2016) Molecular mechanisms of PPARgamma governing MSC osteogenic and adipogenic differentiation. Curr. Stem Cell Res. Ther. 11, 255–264 27 Bionaz, M. et al. (2015) Transcription adaptation during in vitro adipogenesis and osteogenesis of porcine mesenchymal stem cells: dynamics of pathways, biological processes, up-stream regulators, and gene networks. PLoS One 10 e0137644 28 Luo, X. et al. (2008) Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects. Lab. Invest. 88, 1264–1277 29 Song, M. et al. (2015) Genistein exerts growth inhibition on human osteosarcoma MG-63 cells via PPARgamma pathway. Int. J. Oncol. 46, 1131–1140
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30 Lu, Y. et al. (2018) Oridonin exerts anticancer effect on osteosarcoma by activating PPAR–gamma and inhibiting Nrf2 pathway. Cell Death Dis. 9, 15 31 Khalid, A.B. and Krum, S.A. (2016) Estrogen receptors alpha and beta in bone. Bone 87, 130–135 32 Krum, S.A. (2011) Direct transcriptional targets of sex steroid hormones in bone. J. Cell. Biochem. 112, 401– 408 33 Lillo Osuna, M.A. et al. (2019) Activation of estrogen receptor alpha by decitabine inhibits osteosarcoma growth and metastasis. Cancer Res. 79, 1054 34 Lee, C.H. et al. (2012) Ugonin K-stimulated osteogenesis involves estrogen receptor-dependent activation of non-classical Src signaling pathway and classical pathway. Eur. J. Pharmacol. 676, 26–33 35 Pang, X.G. et al. (2018) Quercetin stimulates bone marrow mesenchymal stem cell differentiation through an estrogen receptor-mediated pathway. Biomed. Res. Int. 2018, 4178021 36 Nikitovic, D. et al. (2016) Parathyroid hormone/ parathyroid hormone-related peptide regulate osteosarcoma cell functions: Focus on the extracellular matrix (Review). Oncol. Rep. 36, 1787–1792 37 Carpio, L. et al. (2001) Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways. Am. J. Physiol. Endocrinol. Metab. 281, E489–E499 38 Tashjian, A.H., Jr and Goltzman, D. (2008) On the interpretation of rat carcinogenicity studies for human PTH (1-34) and human PTH(1-84). J. Bone Miner. Res. 23, 803–811 39 Ho, P.W. et al. (2015) Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo. Oncogene 34, 2922–2933 40 Zhang, N. et al. (2014) Hyperoside, a flavonoid compound, inhibits proliferation and stimulates osteogenic differentiation of human osteosarcoma cells. PLoS One 9 e98973 41 Jung, C.H. et al. (2012) Alpinia officinarum inhibits adipocyte differentiation and high-fat diet-induced obesity in mice through regulation of adipogenesis and lipogenesis. J. Med. Food 15, 959–967 42 Liu, C. et al. (2017) Galangin inhibits human osteosarcoma cells growth by inducing transforming growth factor-beta1-dependent osteogenic differentiation. Biomed. Pharmacother. 89, 1415–1421 43 Verrecchia, F. and Redini, F. (2018) Transforming growth factor-beta signaling plays a pivotal role in the interplay between osteosarcoma cells and their microenvironment. Front. Oncol. 8, 133 44 Geng, S. et al. (2014) Coleusin factor, a novel anticancer diterpenoid, inhibits osteosarcoma growth by inducing bone morphogenetic protein-2-dependent differentiation. Mol. Cancer Ther. 13, 1431–1441 45 Chang, Y. et al. (2015) Bufalin inhibits the differentiation and proliferation of cancer stem cells derived from primary osteosarcoma cells through Mir-148a. Cell Physiol. Biochem. 36, 1186–1196 46 Lu, Y. et al. (2018) GLP2 promotes directed differentiation from osteosarcoma cells to osteoblasts
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and inhibits growth of osteosarcoma cells. Mol. Ther. Nucleic Acids 10, 292–303 Valenti, M.T. et al. (2014) Ascorbic acid induces either differentiation or apoptosis in MG-63 osteosarcoma lineage. Anticancer Res. 34, 1617–1627 Huang, X.C. et al. (2014) Synergistic effects of arsenic trioxide combined with ascorbic acid in human osteosarcoma MG-63 cells: a systems biology analysis. Eur. Rev. Med. Pharmacol. Sci. 18, 3877–3888 Ratti, C. et al. (2017) Trabectedin overrides osteosarcoma differentiative block and reprograms the tumor immune environment enabling effective combination with immune checkpoint inhibitors. Clin. Cancer Res. 23, 5149–5161 Gobin, B. et al. (2015) BYL719, a new alpha-specific PI3K inhibitor: single administration and in combination with conventional chemotherapy for the treatment of osteosarcoma. Int. J. Cancer 136, 784–796 Zhang, Y. et al. (2013) All-trans retinoic acid potentiates the chemotherapeutic effect of cisplatin by inducing differentiation of tumor initiating cells in liver cancer. J. Hepatol. 59, 1255–1263 Barroga, E.F. et al. (1999) Effects of vitamin D and retinoids on the differentiation and growth in vitro of canine osteosarcoma and its clonal cell lines. Res. Vet. Sci. 66, 231–236 Zhang, W. et al. (2017) Uev1A facilitates osteosarcoma differentiation by promoting Smurf1-mediated Smad1 ubiquitination and degradation. Cell Death Dis. 8 e2974 Dong, J. et al. (2016) GLIPR1 inhibits the proliferation and induces the differentiation of cancer-initiating cells by regulating miR-16 in osteosarcoma. Oncol. Rep. 36, 1585–1591 Gong, W. et al. (2016) Meteorin-like shows unique expression pattern in bone and its overexpression inhibits osteoblast differentiation. PLoS One 11 e0164446
Yingqian Chen1 Ji Cao1 Ning Zhang2 Bo Yang1 Qiaojun He1 Xuejing Shao1,* Meidan Ying1,* 1 Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China 2 Department of Orthopedics, The Second Affiliated Hospital of Zhejiang University, Hangzhou, China
*Corresponding authors.
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feature Advances in differentiation therapy for osteosarcoma Yingqian Chen1, Ji Cao1, Ning Zhang2, Bo Yang1, Qiaojun He1, Xuejing Shao1, [email protected] and Meidan Ying1, [email protected]
Differentiation therapy: from leukemia to osteosarcoma The concept of differentiation therapy was first introduced during the 1970s, as a means of inducing tumour cells to undergo terminal differentiation. The irreversible changes in the tumour phenotype make it possible to cure the cancer. Currently, differentiation therapy is mainly applied to haematological tumours. One typical example is the combination of retinoic acid (RA) and arsenic for acute promyelocytic leukemia (APL), which has become the standard clinical therapy for this disease. However, the application of differentiation therapy for solid tumours is lagging because of the complexity of the differentiation pathway. It is increasingly believed that solid tumours could also be treated with differentiation therapy, given a better understanding of the details and regulation of the differentiation process. Progress has been
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made with the use of differentiation therapy for solid tumours, especially osteosarcoma, as we discuss here. Osteosarcoma is the most common malignant primary bone tumour in adolescents and children, and is usually accompanied by lung metastasis. Osteosarcoma originates systematically from mesenchymal stem cells (MSCs) or committed osteoblasts and can be divided into osteoblastic, chondroblastic, and fibroblastic subtypes according to the predominantly differentiated component. In addition, osteosarcoma has significant heterogeneity because of its histological heterogeneity and instability of its genetic makeup [1]. Current treatment involves multidisciplinary treatment with neoadjuvant chemotherapy followed by surgery. However, clinical treatment faces numerous difficulties, including the adverse effects of chemotherapies, chemoresistance, and recurrence [2,3]. To overcome
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Differentiation therapy involves the use of agents that can induce differentiation in cancer cells, with the irreversible loss of tumour phenotype. The application of differentiation therapy in osteosarcoma has made progress because of a better understanding of the potential links between differentiation defects and tumorigenesis. Here, we review recent studies on differentiation therapy for osteosarcoma, describing a variety of differentiation inducers. By highlighting these examples of drug-induced osteosarcoma cell differentiation, we can acquire unique insights into not only osteosarcoma treatment, but also novel approaches to transform differentiating drugs into more effective therapies for other solid tumours.
these challenges, more effective treatment options are urgently needed. Given that most osteosarcomas are poorly differentiated histopathologically, differentiation therapy is viewed as a challenge in osteosarcoma treatment research.
Osteogenic differentiation defects The heterogeneous nature of osteosarcoma manifests by varying degrees of mesenchymal differentiation [4]. Moreover, osteosarcoma cells are considered to share similarities with primitive osteoblasts because of their undifferentiated or poorly differentiated stage and their self-renewal and proliferation abilities [5]. In particular, the more malignant phenotypes of osteosarcoma are often associated with characteristics of early osteogenic progenitors [6], suggesting that osteosarcoma is a differentiation dysfunction associated with osteogenic differentiation, which
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might have a role in osteosarcoma development and progression.
morphogenetic proteins (BMPs) [10] S100A4 [11], and S100A6 [12], are also involved. Some of these factors have become therapeutic markers for delineating the effects of differentiation therapy, including CTGF, Osterix, Runx2, osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP), and type 1 collagen [7]. In addition, proteins have been identified as differentiation markers, such as Wnt inhibitory factor 1 (WIF1) [13] and AT-rich sequencebinding protein 2 (SATB2) [14]. These molecules are key to understanding the cause of osteosarcoma and opening the door to differentiation therapy.
Osteogenic differentiation MSCs are bone marrow stromal cells that have the ability to proliferate, self-renew, and differentiate into mature tissues via osteogenic, chondrogenic, adipogenic, and myogenic differentiation [1]. Bone formation mainly refers to the process by which MSCs differentiate into osteoblasts, and the study of osteogenic differentiation is the most extensive and, thus, is our focus here. Osteogenic differentiation is an important process involved in bone formation, growth, and development. It is a well-coordinated multistep process that is regulated by a complex molecular framework [7]. Several transcription factors are involved, including Osterix, Runx2, Sox2, and Sox9 [8]. Other factors include secreted growth factors and their receptors, such as connective tissue growth factor (CTGF) and insulin-like growth factor I (IGF I). IGF I signalling is involved in normal bone growth and differentiation, and the mutations have been identified in IGF signalling genes in 7% of patients [9]. Bone formation-related proteins, such as bone
Associations between osteogenic differentiation defects and osteosarcoma
Differentiation therapy for osteosarcoma
A balance between the differentiation and proliferation potential of stem cells has been proposed [7]. When cells undergo differentiation, they lose their ability to proliferate, and are less sensitive to growth factors [15]. The pathogenesis of osteosarcoma occurs when abnormal osteoblast differentiation is disrupted by mutations or other causes and the cells maintain different stages of primitive osteoblasts. Growth
Features PERSPECTIVE
PD-1/PD-L1
IGF
Interleukin
ra
tio
n
PI3K/AKT/mTOR
Pr
ol
ife
VEGFR
Bo
MMP En ne vi Im TNF-α ro m nm un en e ... t
Ce
ll
PDGFR
By defining osteosarcoma as a disease of differentiation dysfunction, we not only understand the pathogenesis of osteosarcoma from a new perspective, but also provide ideas for new therapeutics for osteosarcoma differentiation. Recent studies have focussed on overcoming the differentiation defects associated with osteosarcoma for therapeutic purposes. Examples of differentiation therapies are described below and summarized in Table 1.
Immunomodulators
Tyrosine kinase inhibitors TGF-β
factors then stimulate the precursor cells to proliferate uncontrollably, resulting in cancer development. Furthermore, osteosarcoma bears incomplete osteoblast differentiation characteristics, resulting in varying degrees of differentiation delay. In this context, there are close relationships between the defects in osteogenic differentiation and the pathogenesis of osteosarcoma. Other signalling pathways, such as cell proliferation, the bone immune environment, and hormonal milieu, are also involved in the development of osteosarcoma (Fig. 1). Therefore, differentiation therapy aims to induce differentiation by restoring the process of osteogenic differentiation in osteosarcoma cells.
...
M mo ili n eu al
Growth Hormone
re
or
Di ffe
H
SOX9 SATB1
Estrogen ...
Runx2
nt
ia
tio
n
BMP
PTH
PTHrP
SATB1
WNT
PPATγ Osterix Notch ...
Hormone modulators
Differentiation inducers Drug Discovery Today
FIG. 1
The main signaling pathways involved in osteosarcoma development and the potential therapeutic strategies. For definitions, please see the main text.
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DRUDIS-2536; No of Pages 8 Drug Discovery Today Volume 00, Number 00 September 2019
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TABLE 1
Summary of potential differentiation inducers in human osteosarcoma. Nuclear receptor agonists RARa agonists (ATRA etc.)
PPARg agonists
Hormone receptor modulators Estrogen receptor agonists PTH/ PTHrP and PTHR1 antagonists
Natural medicines Hyperoside Galangin Coleusin factor Oridonin Bufalin Other inducers GLP2 AsA/ascorbate
Trabectedin Alpelisib (BYL719)
Possible mechanisms
Underlying therapeutic strategies
Refs
Activates RARa; crosstalk with other pathways (MAPKs, NF-kB, TGF-b, Notch, Smad); inhibits M2 polarization of TAMs
RARa-based synergistic strategies: proteasome inhibitor; MDM2 enzyme activity inhibitor HLI373; sumoylation activator. Other synergistic strategies: PPARg agonists; methotrexate Synthetic compounds (thiazolidinedione, troglitazone, ciglitazone, pioglitazone); natural compounds (genistein, oridonin)
[6,17,20–24]
Activates PPARg; bypasses BMP pathway defects
Activates BMP pathway; promotes ER expression Regulates osteogenic differentiation: regulates the oncoprotein c-fos; inhibits PTHR1 pathway
DNA methylation inhibitor (decitabine); natural compounds (ugonin K, quercetin) PTH/ PTHrP: knockout or inhibition of PTHR1; Ezh2 inhibitors
[32–35]
Activates BMP-2 and TGF-b pathways; induces G0/G1 cell cycle arrest Activates (TGF)-b1, Smad2/3 pathway Activates BMP-2 pathway Activates PPAR-g; inhibits Nrf2 pathway Targets miR-148a; regulates DNMT1 and p27
N/A
[40]
N/A N/A N/A N/A
[41,42] [44] [30] [45]
N/A
[46]
Combined with As2O3
[47,48]
Combined with PD-1inhibitors
[49]
Combined with conventional chemotherapy
[50]
Inhibits proliferation rate; inhibits NF-kB; decrease c-Myc, PKM2, CyclinD1 Increases RUNX2 and SPP1 at low concentrations; induces apoptosis at higher concentrations through p21 Favours recruitment of Runx2; reduces proliferation rate Decreases cell proliferation by blocking cell cycle; affects osteoblast and osteoclast differentiation
Nuclear receptor agonists The nuclear receptor superfamily is a group of ligand-activated transcription factors, including retinoic acid receptor a (RARa) and peroxisome proliferation-activated receptor g (PPARg). RARa and PPARg agonists can regulate the terminal differentiation of osteosarcoma.
RARa agonists
[5,6,27–29]
RARa has a pivotal role in many differentiation processes by activating the transcription of downstream differentiation-related genes when binding to RA. All-trans retinoic acid (ATRA) is a vitamin A derivative with an important role in many processes, such as cell differentiation and embryo development [16]. The rapid clearance of APL in response to ATRA was a successful result of differentiation therapy. ATRA promotes osteogenic differentiation of osteosarcoma cells both in vitro and in vivo [17–19]. These results suggest ATRA to be a viable target to develop osteosarcoma treatment. RARa has a crucial role in ATRA-induced differentiation of osteosarcoma cells, and
researchers found phosphorylation of serine 77 and SUMO-1 modification of lysine 399 of RARa to be important for ATRA-induced cell differentiation [17,20]. In addition, ATRA can also crosstalk with other signalling pathways, such as the mitogen-activated protein kinase (MAPK), nuclear factor (NF)-kB, transforming growth factor (TGF)-b, Notch, and Smad signalling pathways [21]. ATRA also inhibits osteosarcoma cell metastasis by inhibiting the M2 polarization of tumour-associated macrophages(TAMs) [22]. These results provide a comprehensive understanding of the specific mechanisms of ATRA in the treatment of osteosarcoma. Although ATRA showed significant effects in preclinical studies in inducing osteosarcoma differentiation, its effects in patients with osteosarcoma have not yet been assessed (Table 2). This might be because the sensitivity of osteosarcoma cells to ATRA and the specificity of treatment remain to be elucidated. Recent studies attempted to further increase the sensitivity of osteosarcoma cells to ATRA and
[37–39]
improve its effectiveness in patients with osteosarcoma. Previous studies found that RARs undergo ubiquitination-mediated degradation, which could affect the ATRA-mediated transcriptional regulation of downstream genes. Therefore, low expression of RARa might be a key factor affecting the sensitivity of the clinical differentiation of osteosarcoma. Thus, attempts have been made to improve the differentiation treatment effect of ATRA by regulating the ubiquitination-mediated degradation of RARa. The transcription factor E2F1 and E3 enzyme MDM2 affect the stability of RARa, attenuating the osteogenic differentiation of osteosarcomas induced by ATRA [23]. Mechanistic studies revealed that MDM2 specifically regulates the degradation of RARa protein and the MDM2 enzyme inhibitor HLI373 effectively promotes osteosarcoma differentiation [24]. In addition to ubiquitination modification, the sumoylation of RARa is also important for its protein stability and protein nuclear localisation. It is speculated that the activation of sumoylation modification
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3
Features PERSPECTIVE
Differentiation agents
Features PERSPECTIVE IUPAC name
Structure
Clinical trials and preclinical models
Refs
Clinical trial
PDX/PDC
Cell lines/ xenografted animals
Cells lines
3,7-dimethyl-9-(2,6,6-trimethyl-1cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid
N/A
PDC
U2OS 143B, K7M2
U2OS
[17,21–24]
Trabectedin
Spiro [6,16-(epithiopropanoxymethano)7,13-imino-12H-1,3-dioxolo [7,8]isoquino [3,2-b] [3]benzazocine-20,1'(2'H)isoquinolin]-19-one,5-(acetyloxy)3',4',6,6a,7,13,14,16-octahydro-6',8,14trihydroxy-7',9-dimethoxy-4,10,23trimethyl-,(1'R,6R,6aR,7R,13S,14S,16R)-
Osteosarcoma, phase 2 (NCT00005625)
PDC
N/A
Saos-2
[49]
Decitabine
5-Aza-2'-deoxycytidine
Sarcoma, phase 1 (NCT02959164, NCT01241162)
N/A
143B
143B
[33]
AsA/ascorbate
Piperazine, 1-((1-phenylcyclohexyl)acetyl)4-(2-phenylethyl)-, coMpd. with L-ascorbic acid
Sarcoma, phase 2 (NCT03468075)
N/A
N/A
MG-63
[47,48]
Alpelisib (BYL719)
(2S)-N1-[4-Methyl-5- [2-(2,2,2-trifluoro-1,1dimethylethyl)-4-pyridinyl]-2-thiazolyl]-1,2pyrrolidinedicarboxamide
N/A
N/A
HOS, MOS-J
HOS, MG-63
[50]
Troglitazone
5-(4-((6-hydroxy-2,5,7,8tetramethylchroman-2-yl)methoxy)benzyl) thiazolidine-2,4-dione
N/A
N/A
N/A
143B, MG63
[5,15]
5-(4-((1-methylcyclohexyl)methoxy)benzyl) thiazolidine-2,4-dione
N/A
N/A
N/A
143B, HOS
[5]
Approved drugs ATRA
Agents in development Ciglitazone
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Differentiation inducers: clinical trials and preclinical models in osteosarcoma.
PERSPECTIVE
4
TABLE 2
IUPAC name
Structure
Clinical trials and preclinical models
Refs
Clinical trial
PDX/PDC
Cell lines/ xenografted animals
Cells lines
5-((3S,5R,8R,9S,10S,13R,14S,17R)-3,14dihydroxy-10,13-dimethylhexadecahydro1H-cyclopenta [a]phenanthren-17-yl)-2Hpyran-2-one
N/A
PDC
N/A
MG-63
[45]
Oridonin
(14 r)-7-alpha,20-epoxy-1-alpha,6beta,7,14-tetrahydroxykaur-16-en-15-one
N/A
N/A
HOS
MG-63 HOS, Saos-2, U2OS
[30]
Galangin
3,5,7-trihydroxy-2-phenyl-4h-benzopyran4-on
N/A
N/A
MG-63
U2OS, MG-63
[41,42]
Genistein
5,7-dihydroxy-3-(4-hydroxyphenyl)-4Hchromen-4-one
N/A
N/A
N/A
MG-63
[29]
Quercetin
4H-1-benzopyran-4-one, 2-(3,4dihydroxyphenyl)-3,5,7-trihydroxy-
N/A
N/A
N/A
Mouse BMSC
[35]
Hyperoside
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl) oxy)-4H-chromen-4-one
N/A
N/A
N/A
U2OS, MG-63
[40]
Coleusin factor GLP2
N/A N/A
N/A N/A
N/A N/A
MG-63 MG-63
U2OS, MG-63 MG-63
[44] [46]
N/A N/A
5 Features PERSPECTIVE
PERSPECTIVE
Bufalin
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TABLE 2 (Continued )
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of RARa could regulate the therapeutic effects of tumour differentiation. Several therapeutic strategies can be proposed for tumour differentiation based on the synergy between RARa post-translational modification regulation and ATRA: the proteasome inhibitor bortezomib, the MDM2 enzyme activity inhibitor HLI373, and a sumoylation activator could be used as ATRA sensitisers. In addition, RARa/MDM2/E2F1 could be a molecular marker for the treatment of osteosarcoma via differentiation therapy. Most of these outcomes are indicated by preclinical models but not yet by clinical trials, although this might only be a matter of time. ATRA can also synergize with agents to promote differentiation treatment effects, such as PPARg agonists [6] and methotrexate [25]. The latter is an antineoplastic agent that is currently in clinical trials for osteosarcoma treatment. It might be that ATRA would be a treatment for patients with osteosarcoma treated with highdose methotrexate (HDMTX).
PPARg agonists
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PPARg belongs to a nuclear receptor superfamily of PPARs and has an important role in osteogenic and adipogenic differentiation, cell proliferation, and apoptosis [26,27]. Exogenous expression of PPARg2, RXRa, and RARa, individually or in combination, inhibits in vivo osteosarcoma growth [6]. Additionally, some PPARg agonists can inhibit the proliferation of osteosarcoma cells and promote their differentiation in vitro, including thiazolidine derivatives, such as troglitazone and ciglitazone [5], which are currently used as insulin-sensitizing agents for antidiabetic drugs. PPARg agonists can bypass the defects in the differentiation pathway regulated by BMPs in osteosarcoma cell lines [28]. Some natural compounds can also promote the expression of PPARg and have a therapeutic effect, such as genistein [29] and oridonin [30]. Thus, the use of PPARg agonists is a good potential synergistic strategy for ATAR treatment.
Hormone receptor modulators Estrogen receptor agonists Estrogen is an important steroid hormone that is mediated by estrogen receptors (ER). Estrogen can regulate osteogenic differentiation and bone growth, facilitating bone formation and maintaining bone mineral density [31]. It can also induce the expression of genes related to osteogenic differentiation, such as those encoding BMPs and ALP [32]. In addition, the expression of ERa in osteosarcoma is significantly decreased 6
compared with that in normal tissues. Recently, the DNA methylation inhibitor decitabine was found to inhibit osteosarcoma cell proliferation and promote osteogenic differentiation via inducing ERa expression [33]. In addition, the natural compounds, ugonin K [34] and uercetin [35], were reported to have a role in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by acting in an ERdependent manner. These studies demonstrate the role of estrogen in regulating normal bone development and show that ER modulators act as corresponding differentiation inducers.
Parathyroid hormone and PTHR1 antagonists Parathyroid hormone (PTH) is a peptide hormone secreted by the parathyroid gland. PTHrelated protein (PTHrP) is similar in amino acid sequence to PTH. Both can bind to, and activate, PTH receptor 1 (PTHR1) to regulate bone differentiation and development and calcium homeostasis [36]. Both PTH and PTHrP can promote the differentiation of osteosarcoma cells [37] However, preclinical toxicology studies showed that a high incidence of osteosarcoma occurred with long-term use and high doses, but not with therapeutic doses of PTH administered to rats [38]. The correlation between PTH and osteosarcoma is intriguing. Recent studies found that knockout of PTHR1 affected osteosarcoma growth and promoted differentiation, whereas overexpression of PTHR1 had the opposite effect. This process was mediated by polycomb repressive complex 2 (PRC2), a finding supported by using Ezh2 inhibitors [39]. These studies suggest that inhibition of PTHR1 signalling is a useful way to increase differentiation and that PTHR1 antagonists are a potential treatment for osteosarcoma; however, the role of PTH in osteosarcoma requires further research.
Other potential differentiation inducers for osteosarcoma Natural compounds Many studies found that Chinese compounds can promote differentiation in osteosarcoma cells. For example, the flavonoid hyperoside, the main pharmacologically active compound in Hypericum perforatum, stimulates osteoblastic differentiation by activating the BMP-2 and TGFb pathways and inducing G0/G1 cell cycle arrest [40]. Galangin, the main active ingredient of Alpinia officinarum, activates the TGF-b1/ Smad2/3 signalling pathway [41,42]. With both these drugs, the antitumour effects are promoted by activating TGF-b to promote tumour
differentiation. Given the complicated role of TGF-b, such as its role in tumour metastasis, it cannot be defined as a therapeutic target, and further research is required [43]. Other flavonoids, such as ugonin K [34], quercetin [35], and genistein [29], have been verified as potential differentiation inducers for osteosarcoma. Likewise, the effects of terpenoids, including coleusin factor and oridonin, have been reported recently. Coleusin factor can inhibit the growth of osteosarcoma cells both in vitro and in vivo by activating the BMP-2 pathway [44]. [30] Finally, bufalin was found to inhibit the stemness and cell proliferation of osteosarcoma stem cells by directly targeting miR-148a, thereby regulating DNA methyltransferase 1 and p27 [45].
Other types of inducers In addition to natural medicines, other types of agent have clear effects on the differentiation of osteosarcoma cells, including peptides, and drugs currently used for other purposes. For example, glucagon-like peptide 2 (GLP2), a proglucagon-derived peptide, inhibits the growth of osteosarcoma cells and promotes the osteogenic differentiation of osteosarcoma cells by inhibiting NF-kB and decreasing levels of cMyc, PKM2, and CyclinD1 [46]. In addition, ascorbic acid (AsA)/ascorbate induces differentiation by increasing Runx2 and SPP1 gene expression and, at higher concentrations, it induces apoptosis through p21 in poorly differentiated osteosarcoma cells [47]. Huang et al. revealed the synergistic mechanism of As2O3 and AsA in human osteosarcoma [48]. Similarly, trabectedin recruits Runx2 and induces the striking differentiation of osteosarcoma cells. Combining trabectedin with immune checkpoint inhibitors and the accompanying enhanced expression of the inhibitory checkpoint molecule PD-1 has been suggested [49]. BYL719 (alpelisib) is a new a-specific PI3K inhibitor with dual functions in osteoblast and osteoclast differentiation and has been suggested to be combined with conventional chemotherapy for the treatment of osteosarcoma [50]. Thus, although these compounds or drugs were developed for other indications, they have been found to be useful as differentiation inducers for osteosarcoma.
Moving forward with differentiation therapy
Therapeutic potential in osteosarcoma Differentiation inducers for osteosarcoma have made significant progress in preclinical studies and some drugs approved for other indications have been the focus of clinical trials, such as
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Differentiation therapy and tumour heterogeneity Tumour heterogeneity makes it difficult to treat tumours, particularly osteosarcoma. Numerous studies have shown that ATRA can induce tumour differentiation in various cancers, including osteosarcoma, leukaemia, and neuroblastoma, which are paediatric cancers with obvious differentiation defects. Therefore, differentiation therapy might also induce the differentiation of different subtypes of osteosarcoma. Earlier studies reported that ATRA can induce different types of osteosarcoma, including fibroblast, chondroblast osteoblastic, and undifferentiated types [52]. Therefore, dif-
ferences in the classification of osteosarcoma might not affect the efficacy of differentiation therapy. However, the heterogeneous expression of differentiation therapeutic targets can affect the sensitivity of differentiation therapy. For example, RARa expression can lead to differences in the sensitivity of osteosarcoma cells to ATRA [23]. These results suggest that we need to determine the appropriate treatment population based on the therapeutic targets of differentiation therapy, similar to the concept of biomarkers. Therefore, as long as there are effective differentiation inducers and suitable biomarkers present, differentiation therapy will be more able to exert its effects in heterogeneous osteosarcoma or other paediatric cancers characterized by cellular differentiation. In recent years, there has been some progress in this area of research. For example, the regulatory mechanisms of osteogenic differentiation have been further elaborated, with various proteins revealed to be directly or indirectly involved in osteogenic differentiation and associated with the development of osteosarcoma. For example, Uev1A was reported to promote osteogenic differentiation of osteosarcoma by regulating the ubiquitination of the osteosarcoma-promoting factor Smad1. Uev1A overexpression not only promoted osteogenic differentiation of osteosarcoma, but also increased the sensitivity of osteosarcoma to chemotherapy [53]. In addition, human glioma pathogenesis-related protein 1 (GLIPR1) [54] and Meteorin, glial cell differentiation regulator-like (METRNL) [55] have both been reported to regulate the differentiation of osteosarcoma. Even though differentiation therapy for osteosarcoma has unique advantages and great clinical potential, it is progressing slowly. The crux of the problem lies in that there are too few known differentiation inducers, with little research on targets, especially for osteosarcoma. Moreover, in the context of the heterogeneity of osteosarcoma, there is a lack of biomarkers to determine the appropriate sensitive population for differentiation therapy. Thus, there is a need to use targeted ideas to study differentiation therapy in the future.
Concluding remarks Here, we have provided theoretical and preclinical evidence for the use of differentiation therapy for osteosarcoma. The discovery of molecules that can induce osteosarcoma to undergo terminal differentiation in vitro has promoted the use of differentiation therapy against osteosarcoma, with significant improvements in the treatment of solid
tumours. Researchers still have a long way to go to discover more effective differentiation inducers and biomarkers, as well as the indepth exploration of the molecular mechanisms involved. If osteosarcoma-induced differentiation mechanisms and regulatory factors can be elucidated, more differentiation-inducing agents and differentiation-inducing protocols can be proposed, rendering differentiation therapy a powerful weapon in the treatment of osteosarcoma. Differentiation therapy will also be an effective strategy for other solid tumours.
Acknowledgements This work was supported by the State Key Program of the National Natural Science Foundation of China (No. 81830107 to Q. He) and a grant from the National Natural Science Foundation of China (No. 81803552 to X.S.). We thank Chenghao Pan for help with drawing the chemical structures used in the figures. References 1 Mutsaers, A.J. and Walkley, C.R. (2014) Cells of origin in osteosarcoma: mesenchymal stem cells or osteoblast committed cells? Bone 62, 56–63 2 Isakoff, M.S. et al. (2015) Osteosarcoma: current treatment and a collaborative pathway to success. J. Clin. Oncol. 33, 3029–3035 3 Kansara, M. et al. (2014) Translational biology of osteosarcoma. Nat. Rev. Cancer 14, 722 4 Adamopoulos, C. et al. (2016) Deciphering signaling networks in osteosarcoma pathobiology. Exp. Biol. Med. 241, 1296–1305 5 Haydon, R.C. et al. (2007) Osteosarcoma and osteoblastic differentiation: a new perspective on oncogenesis. Clin. Orthop. Relat. Res. 454, 237–246 6 He, B.C. et al. (2010) Synergistic antitumor effect of the activated PPARgamma and retinoid receptors on human osteosarcoma. Clin Cancer Res. 16, 2235–2245 7 Wagner, E.R. et al. (2011) Defective osteogenic differentiation in the development of osteosarcoma. Sarcoma 2011, 325238 8 Almalki, S.G. and Agrawal, D.K. (2016) Key transcription factors in the differentiation of mesenchymal stem cells. Differentiation 92, 41–51 9 Behjati, S. et al. (2017) Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma. Nat. Commun. 8, 15936 10 Bragdon, B. et al. (2011) Bone morphogenetic proteins: a critical review. Cell Signal. 23, 609–620 11 Chen, X. et al. (2013) The E-F hand calcium-binding protein S100A4 regulates the proliferation, survival and differentiation potential of human osteosarcoma cells. Cell. Physiol. Biochem. 32, 1083–1096 12 Li, Y. et al. (2015) The calcium-binding protein S100A6 accelerates human osteosarcoma growth by promoting cell proliferation and inhibiting osteogenic differentiation. Cell. Physiol. Biochem. 37, 2375–2392 13 Baker, E.K. et al. (2015) Wnt inhibitory factor 1 (WIF1) is a marker of osteoblastic differentiation stage and is not silenced by DNA methylation in osteosarcoma. Bone 73, 223–232
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Features PERSPECTIVE
trabectedin, decitabine and ascorbate (Table 2). Given this progress, other approved drugs with appropriate preclinical reactions, such as RA and alpelisib, should be progressed to clinical trials in patients with osteosarcoma as soon as possible. What is the way forward for differentiation therapy in the treatment of osteosarcoma? Osteosarcoma is a highly aggressive cancer for which treatment has remained essentially unchanged for >30 years, with numerous problems of drug resistance, recurrence, and adverse effects of chemotherapy, suggesting that new therapies would be well received. In this context, targeted therapy and immunotherapy have attracted much attention in clinical and preclinical studies, and differentiation therapy, with its unique features, is another important choice for osteosarcoma treatment for several reasons: (i) it tends to be less toxic compared with conventional cancer treatments. Most differentiation inducers are physiological substances in the body, such as ATRA and ascorbic acid, and do not have obvious adverse effects on the body; (ii) given its unique ability to induce tumour cell differentiation, especially of cancer stem cells, differentiation therapy could be used for synergistic application with other therapies including chemotherapy, targeted therapy, and immunotherapy, to improve complete remission and cure rates. For example, ATRA can potentiate the effect of chemotherapeutic drugs, such as cisplatin and methotrexate [25,51]; and (iii) differentiation therapy can also activate antitumour immune responses, such as by inhibiting the M2 polarization of TAMs [22], thereby promoting immunotherapy effects. Therefore, as a new approach to the treatment of osteosarcoma that has low toxicity, differentiation therapy can provide a synergistic strategy with existing treatment systems, and has the potential to improve the survival rate of patients.
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Yingqian Chen1 Ji Cao1 Ning Zhang2 Bo Yang1 Qiaojun He1 Xuejing Shao1,* Meidan Ying1,* 1 Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China 2 Department of Orthopedics, The Second Affiliated Hospital of Zhejiang University, Hangzhou, China
*Corresponding authors.
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