Growth factors, cytokines, and pediatric malignant primary bones tumors

Introduction

Primary bone tumors constitute a heterogeneous family of rather rare neoplasms corresponding to oncogenic conversion of cells from the lineages implicated in bone formation and homeostasis: the osteoblastic, the chondroblastic, and the osteoclastic lineages. Part of the primary bone tumors belongs to the pediatric neoplasms and two of those are particularly malignant, namely the osteosarcoma (OS) and the Ewing's sarcoma (ES). The oncogenic event leading to OS occurrence is still elusive and, similarly to many tumors, mutations in P53 , Rb , and Mdm2 genes have been reported [ ]. In contrast, ES incidence is associated to chromosomes rearrangements (mainly translocation between chromosomes 11 and 22) leading to the synthesis of an aberrant fusion protein (transcription factor) encoded by the first exons of the EWS gene and the last exons from a gene of the ETS family, the most frequent being the EWS-FLI1 fusion protein [ ]. ES as OS are highly malignant tumors with a destruction of the adjacent bone through the activation of osteoclasts, the formation of an anarchic tumoral osteoid tissue and an important metastatic dissemination potential mainly toward other bone sites and the lung with formation of mineralized nodules [ ]. Actual management of patients with ES and OS, taking into account the low efficiency of radiotherapy in the bone microenvironment, is a three-step protocol consisting in a preoperative conventional chemotherapy to induce necrosis in the tumor and clarified the tumor's margins, a surgical resection of the tumor and a postoperative chemotherapy to target potential remnant tumor cells [ ]. Such protocol enables to reach a 5-year recurrence-free incidence higher than 70%. However, if metastases are present at diagnosis or in the case of resistance to the chemotherapy, this incidence dramatically down under the 30%. In the last decades many research studies have been devoted to the identification of new therapeutic targets implicated in either the metastatic dissemination or the chemotherapeutic resistance of OS and ES (in addition to part in the tumor growth) in order to ameliorate the life span of patients.

Considering pediatric tumors, the potential implications of growth factors known to play a part in the physiological skeleton growth were logically investigated as was the potential roles of cytokines implicated in the bone modeling and remodeling. This chapter aims to review the knowledge on the implications of four families of growth factors (Wingless (WNT), fibroblast growth factor (FGF), Hedgehog (HH), and transforming growth factor (TGF) families) and one family of cytokines (Interleukin 6 (IL6)) in the ES and OS growth, metastatic disseminations, and chemotherapeutic resistances.

TGF-β family implications in pediatric malignant primary bone tumors

The TGF-β family is composed of 33 members including the TGF-βs, activins/inhibins, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and Nodal/left-right determination factors [ ]. Indeed, 3 different TGF-βs (TGF-β1, TGF-β2, and TGF-β3), 7 activins/inhibins (ACT-A, ACT-AB, ACT-B and INH-A, INH-BA, INH-BB, INH-BC), 10 BMPs (BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, and BMP-15 (also called GDF-9b)), 10 GDFs (GDF-1, GDF-2 (also called BMP-9), GDF-3, GDF-5 (also called BMP-14), GDF-6 (also called BMP-13), GDF-7 (also called BMP-12), GDF-9, GDF-10 (also called BMP-3b), GDF-11, and GDF-15), and 3 Nodal/left-right determination factors (Nodal, Lefty-1 (also called BMP-17), and Lefty-2) have been identified and constitute this large family.

Members of this superfamily interact as dimers with two types of cell surface receptors (types I and II) that possess intrinsic serine/threonine kinase activities in their intracellular domains ( Table 18.1 ). Functionally, the ligand binds to the type II receptor and initiates a cascade reaction leading to the recruitment, the phosphorylation, and the activation of the type I receptor. This induces the phosphorylation of proteins of the SMAD family ( Table 18.1 ) called the R-SMADs (SMAD-1, SMAD-2, SMAD-3, SMAD-5, and SMAD-8/9) that translocate to the nucleus and interact with SMAD-4 (the unique Co-SMAD) what enable the activation of this transcriptional complex responsible for the expression of a large variety of genes ( Fig. 18.1A ). Another subgroup of SMAD proteins exists called the I-SMADs (SMAD-6 and SMAD-7) that inhibit the R-SMADs.

Concerning the pediatric malignant primary bone tumors, most publications have concerned the implications of TGF-βs and the BMPs proteins in the tumor growth, metastatic dissemination, and resistance to chemotherapies. The expressions of the three TGF-βs were also reported [ ] and mainly associated to the tumor progression [ ] and high-grade tumor classification (TGF-β1; [ , ]). The TGF-βs expression levels in the tumor [ ] and in the blood [ , ] were proposed as prognostic factors before and during the chemotherapy treatment. Polymorphism in the TGF-β1 gene (29T/C) was evidenced to influence the susceptibility to osteosarcoma [ , ]. The expressions of the receptors of the TGF-βs (ALK-4, ALK-5, and TβR-II) were reported [ ] and polymorphisms in the ALK-5 gene [ ] and the TβR-II gene [ ] were also associated to the susceptibility to osteosarcoma. In the ES, the EWS/FLI1 fusion protein was shown to regulate the expression of TβR-II [ ]. The SMAD proteins expression (SMAD-1, SMAD-2, SMAD-4, SMAD-5, and SMAD-7) was also reported as perturbed [ , ]. Concerning the BMPs expression in pediatric malignant primary bone sarcomas, historically the first BMPs have been isolated from osteosarcoma cell cultures in the 1980s based on their ability to induce bone formation [ ], so logically most of BMP family members were further found expressed in these tumors: BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, and BMP-8b [ , ]. Interestingly, to date, a polymorphism increasing the susceptibility to osteosarcoma was only evidenced in the BMP-2 gene [ ]. Regarding the receptors of the BMPs, most were detected in these tumors (BMPR-II [ , ], ALK2 [ ], ALK3 [ ], ALK6 [ ]) suggesting the existence of autocrine and paracrine loops of stimulation. The other members of the TGF-β family expressed in these tumors are the GDF-10 [ ] and the activin A [ , ] both evidencing protumoral effect.

Concerning the TGFs and BMPs effects on the tumor growth, protumoral and antitumoral effects have been reported depending on the growth factor considered and for the same factor on the stage of differentiation of the tumor cell. Indeed, TGF-β1 was evidenced to have a protumoral effect on law differentiated cells by maintaining the undifferentiated status [ , , ] and/or an antitumoral effect on predifferentiated cells boosting the osteoblastic differentiation [ , ] through the stimulation of the expression of bone matrix proteins namely the osteopontin, the bone sialoprotein, the osteocalcin, and the alkaline phosphatase [ ] and the modulation of the PTH/PTHrP hormonal effect [ ].

Several studies have been devoted to decipher the mechanisms of action of TGF-βs in the tumor cells to define therapeutic targets to block the wrong effect of these factors. The TGF-β regulator factor 4 (TBRG4) was recently shown to be able to balance the TGF-β1 protumoral effect [ , ]. Several miRNAs were also implicated in the modulation of the TGF-βs signaling with protumoral (miR-522 [ ]; miR-339-5p [ ]; miR-181c [ ]; miR-20b [ ]; miR-17-92 [ ]) or antitumoral (miR-29 [ ]; miR-34a [ ]; miR-153 [ ]) effects.

Concerning the BMPs, pro- and antitumoral effects were also reported. BMP-2 was the most studied member of the family. Few works have reported a protumoral effect of BMP-2 and such effect was systematically associated to an indirect pathway, for instance, through the stimulation of the Wnt signaling or cross-modulations with the TGF-β signaling [ , ]. The antitumoral effect of BMP-2 was associated to the stimulation of the differentiation of the tumor cells [ ] through the modulation of the expression of the osteoblast master gene Runx2 [ , ], the gene Sox9 driving the chondroid phenotype [ ], the gene Adamts7 [ ], or the gene of the calcitonin-related peptide [ ]. Interestingly such differentiating effect of BMP-2 was also observed in presence of high expression of aldehyde dehydrogenase [ ] or low expression of either miR-29c [ ] or sclerostin [ ]. Only two other BMPs, BMP-7 and BMP-9, were shown to have similar prodifferentiation effect on osteosarcoma [ , , , ].

Concerning the established parts of members of the TGF-β family in the occurrence of metastases [ , , ], four processes have been shown modulated. The first one is the mesenchymal–epithelial transition (MET) that supports the occurrence of metastases. TGF-βs and BMP-2 have been shown to stimulate the MET [ , ] that can be blocked by BAMBI, the BMP and activin membrane-bound inhibitor [ , ].

The second is the stimulation of the neoangiogenesis that favors the metastatic process. TGF-βs and BMP-2 have been associated to such neoangiogenesis stimulation [ , ].

The third is the immune escape that enables the tumor cell to survive during its travel in the blood (for review, see Ref. [ ]).

The fourth is the induction of cell migration, invasion, and extravasation. TGF-βs and BMP-2 have been associated to the stimulation of both invasion and migration through the control of the cellular adhesion [ ], the regulation of the expression of the MMPs [ , , ], the versican [ , ], the hyaluronan [ ], the cysteine-rich protein 61 [ ], the decorin [ ], the S100A4 protein [ ], and the lumican [ ].

Interestingly, the prometastatic effect of BMP-2 was recently shown dependent of the expression levels in the tumor cell of the miR-29c [ ] and the aldehyde dehydrogenase [ ] what open a new window in the therapeutic approach of the primary bone tumor metastatic dissemination. The observation that BMP-9 have an antimetastatic effect through the downregulation of PI3K/AKT signaling [ ] and the decrease expression of MMP-9 is also of promising therapeutic interest.

Concerning the impact of the TGF-β family members on the resistance to chemotherapeutic agents, surprisingly only data on the TGF-βs implications have been published with an expression level in the tumor cell correlated to the resistance and a resensitization that can be achieved by a blockage of the autoregulatory loops [ ] or an inhibition of the TGF-β signaling (Ly2109761 inhibitor [ ]). Mechanistically, such a resistance induction was associated to the modulation of the autophagy [ ] and anoikis [ ] processes, the regulation of miRNAs expression (miR-499a [ ]; miR-202 [ ]), or the dedifferentiation effect that induces a quiescent status not targetable by most antimitotic drugs [ ].

Many synthetic and highly specific inhibitors of the TGF-β family signaling pathways have been generated in the last decades and only few have been evaluated in primary bone tumors. The explanation may be in the paradoxical reverse impacts of the TGF-β family signaling pathways on the tumor growth and metastatic dissemination according to the cellular differentiation stage what make difficult to target so heterogeneous tumors. However and interestingly, old drug and natural compounds have been shown to efficiently target the TGF-β family signaling pathways in pediatric malignant primary bone tumors as the Propofol that block the TGF-β1 effect [ ], the Suramin that block the TGF-β effect [ ], and the Coleusin stimulating the prodifferentiating effect of BMP-2 [ ]. Deeper analyses of these drugs' mechanisms of action in tumor cells may open new perspectives in the targeting of the TGF-β family signaling pathways to treat pediatric malignant bone sarcomas.