Regulated cell death and drug resistance in malignant bone tumors

Introduction

Regulated cell death (RCD) is a physiological cell death program of eukaryotic cells to maintain tissue homeostasis and integrity and involves different mechanisms including apoptosis, autophagic cell death, and necroptosis [ ]. RCD has been extended in recent years to include autophagy and necroptosis, both of which are independent in their signaling mechanisms from apoptosis but equally important for organism development and preservation [ , ]. Damage of the cell death program can lead to tumor formation by shifting the tightly regulated balance between tissue integrity and cell death into the direction of cell growth, and can result in treatment resistance, since radiotherapy and most of chemotherapeutic agents induce RCD.

Apoptosis is an active process in which caspases (CASPs), a family of cysteine proteases, are key mediators [ ]. In humans, there are 14 different caspases known. They are synthesized as inactive proenzymes consisting of a prodomain, a large and small subunit. The active caspases are tetramers composed of two large and two small subunits arising from the cleavage of procaspases. So-called initiator caspases, such as CASP8, CASP9, and CASP10, receive the apoptotic signal, get activated, and in turn activate effector caspases such as CASP3, CASP6, and CASP7. These induce the apoptotic phenotype by cleavage of enzymes such as the DNA-repair enzyme PARP, activation of endonucleases, and cleavage of structural proteins including lamins, which are important for the maintenance of the nuclear membrane. The activity of the caspases is tightly controlled by proteins of the inhibitor of apoptosis family (IAP), including NAIP, BIRC2, BIRC3, XIAP, survivin (BIRC5), BRUCE, BIRC7, and BIRC8 [ , ].

Two major apoptotic pathways exist, the intrinsic and extrinsic pathways [ , ]. A key event in the intrinsic pathway is the disruption of the mitochondrial membrane leading to the release of cytochrome c which binds the cytosolic protein APAF1 (apoptosis activating factor 1). Both proteins together bind pro-CASP9 and form the apoptosome wherein pro-CASP9 is cleaved into active CASP9. Loss of mitochondrial membrane integrity is controlled by a tight balance between pro- and antiapoptotic members of the BCL2 family of proteins [ ]. Twenty members of this family are known, divided into three subfamilies, depending on the presence of conserved BCL2 homology (BH) domains [ ]. The proapoptotic proteins comprise two families, one comprising BAX, BAK1, BAD which contain either three BH homology domains (BH1-3) the other with only one domain (BH-3) such as BID, BCL2L11, BBC3 and PMAIP1. The antiapoptotic family includes BCL2, BCL2L1, BCL2L2, and MCL1. BCL2 and BCL2L1 form stable complexes with the proapoptotic BH-3 domain-only proteins, thereby preventing the activation and translocation of BAX and BAK to the mitochondria. This balance of pro- and antiapoptotic proteins is influenced by TP53 which becomes activated upon cellular stress. TP53 induces transcriptional activation of proapoptotic BAX and PUMA (BBC3), thereby shifting the balanced network of this family of proteins toward apoptosis [ , ]. Chemotherapeutic agents induce intracellular damage. They often induce apoptosis via the intrinsic pathway. Disruption of the intrinsic pathway via mutations in proapoptotic genes, e.g., TP53 , or BAX , or overexpression of antiapoptotic proteins, such as BCL2 or survivin, often leads to chemoresistance.

Binding of death ligands to their receptors activates the extrinsic apoptotic pathway [ , ]. Death receptors belong to the tumor necrosis factor receptor superfamily, including TNFRSF1A (DR1/CD120a/p55/p60), FAS (Apo-1/CD95), DR3 (APO-3/LARD/TRAMP), TRAILR1 (DR4/APO-2), TRAIL2 (DR5), DR6, and ectodysplasin A receptor (EDAR). Activation of these death receptors leads to trimerization and assembly of death-inducing signaling complex (DISC), including the FAS-associated death domain–containing protein (FADD) and the initiator caspases, proCASP8/10 [ , ]. The interaction between FADD and CASP8/10 results in autoproteolytic cleavage and the activation of CASP8/10 [ ]. CASP8 and CASP10 activate effector caspases and the proapoptotic BCL2 family protein BID.

Apoptosis via the extrinsic pathway is tightly controlled and resistance can occur at various levels. On the receptor level there are TRAILR3 and TRAILR4 which do not contain a death domain and can sequester TRAIL from the apoptosis-inducing receptors TRAILR1 and TRAILR2. Overexpression of the protein cFLIP (CFLAR) has been shown to prevent the binding of the adaptor FADD to proCASP8 and proCASP10. Mutations in FAS as well as absent expression of death receptors or CASP8 and CASP10 are also known causes of resistance to the induction of apoptosis via the extrinsic pathway.

Another response to cellular stress besides apoptosis is autophagy . It is a highly conserved process characterized by vesicular sequestration and degradation of cytoplasmic components. Autophagy is induced and precisely regulated following nutrient deprivation, via growth factors, hormones, intracellular energy information, and cell stressors such as hypoxia, osmotic stress, reactive oxygen species (ROS), and viral infection [ ]. Main steps in autophagy are induction, vesicle nucleation and elongation, autophagosome formation, and autolysosome formation. More than 30 autophagy-related genes (ATGs) have been characterized; several molecular complexes seem essential in the process of autophagy [ ]. They are divided into five groups according to their function in the process of autophagy: ATG1 protein kinase complex, class III phosphatidylinositol 3 kinase (PI3K)-Beclin 1 (BECN1) complex, ATG12 conjugation system, ATG8 conjugation system, and ATG9 [ ]. Upon mTOR inhibition, Unc-51-like kinase 1 (ULK1) and ULK2, two mammalian homologs of ATG1, are activated, followed by phosphorylation of FIP200 (a focal adhesion kinase family interacting protein of 200 kDa) and ATG13, which initiate autophagy activity [ ]. The class III PI3 kinase-Beclin complex is essential for vesicle nucleation. It is crucial for recruiting the ATG12-ATG5 conjunction to the preautophagosomal structure. Autophagy can precede apoptosis and serve as an alternative mechanism to activate CASP8 [ ]. Members of the BCL2 family are involved in regulation of autophagy as well. BCL2 can form a complex with BECN1 inhibiting autophagy. Dissociation of this complex initiates autophagy [ , ]. Deregulation of autophagy might contribute to resistance of tumor cells toward environmental stress factors like nutrient deprivation and hypoxia. For example, upregulation of intracellular components such as high mobility group box 1 protein (HMGB1) contributed to chemotherapy resistance [ ]. Drugs inducing autophagy in tumor cells death may complement apoptosis-inducing anticancer agents in order to maximize the cytotoxic effect on a tumor [ ].

Necroptosis has been identified as an independent cell death mechanism. It is triggered by multiple stimulators, and interacts with death receptors (TNFRSF1A, TRAILR, or FAS), toll-like receptors (TLRs), and cellular metabolic and genotoxic stress. The most important signal molecules of necroptosis are the serine/threonine kinase receptor interaction protein 1 (RIP1) and RIP3 as well as the pseudokinase mixed lineage kinase domain-like (MLKL). For example, binding of TNF-α to its receptor TNFRSF1A leads to the internalization of the receptor, the assembly of RIP1 together with RIP3 into the necrosome complex, and the activation of RIP3 [ ]. Then, the activated RIP1 and RIP3 transphosphorylate each other and initiate necroptosis. Both RIP1 and RIP3 necrosome recruits and activates MLKL and phosphoglycerate mutase 5 (PGAM5). MLKL is phosphorylated and multimerized, is inserted into the plasma membrane, and forms channels that increase NA ⁺ influx, osmotic pressure, and membrane rupture, ending with necroptosis-induced cell death [ , ].

Subsequently, the current status of individual therapeutic treatment is correlated to pathways of RCD in malignant bone tumors. Insight into new vulnerabilities discovered is given, new treatment concepts are highlighted, and their potential from the perspective of RCD is discussed.

Osteosarcoma

Osteosarcomas (OSs), especially high-grade OSs (HGOSs) comprising 90% of OSs, are genetically unstable malignancies presenting complex karyotypes with a large variety of numerical and structural variants often associated with chromosomal alterations named kataegis and chromothripsis [ ]. For patients with localized disease standard chemotherapy consists of a three-drug combination with methotrexate, doxorubicin, and cisplatin. This leads to a 5-year survival of approximately 80% in patients with nonmetastatic OS and complete tumor resection [ ]. However, the survival rate of high-risk patients with nonresectable, primary metastatic, or relapsed disease is still inacceptable and requires concerted efforts to improve therapy [ ].

Single-nucleotide polymorphisms (SNPs) in several candidate genes of possible relevance for the biology, treatment response, and drugs activation or detoxification of OS have been evaluated in order to identify markers predictive of tumor progression, therapy response, or patients' susceptibility to develop treatment-related toxicities [ ]. Furthermore, next-generation sequencing (NGS) approaches led to the identification of OS driver genes that may not have emerged with conventional techniques [ , , ].