IRE1 is a type I ER membrane protein present in animals and yeast and is considered to be the protein with the highest degree of evolutionary conservation [29]. IRE1, PERK, and ATF6 contain three structural domains: an ER luminal domain (LD), a single-pass membrane-spanning domain, and a cytosolic domain [30]. The N-terminal LD interacts with BiP and misfolded proteins within the ER and is used to identify misfolded proteins. The IRE1 C-terminal cytoplasmic region influences cell fate through serine/threonine kinase and endoribonuclease (RNase) domains [28,31]. Under severe ERS, the IRE1 kinase structural domain can trigger cell death by mobilizing c-Jun N-terminal kinase (JNK) and, subsequently, proteins from the pro-apoptotic BCL-2 family [31,32,33,34]. The IRE1 RNase structural domain can induce X-box binding protein 1 (XBP1) mRNA splicing and regulate IRE1-dependent decay (RIDD). IRE1 has two distinct isoforms: IRE1α and IRE1β. IRE1α is the major isoform associated with UPR, and both IRE1α and IRE1β perform mRNA splicing and induce RIDD activation, with IREα exhibiting a stronger splicing activity and IRE1β inducing stronger RIDD activation [35,36]. The IRE1 RNase structural domain is activated for the unconventional splicing of XBP1 mRNA in the cytoplasmic matrix, which produces a modified mRNA isoform (XBP1s) encoding the functionally active protein XBP1 (also known as XBP1s). This protein can upregulate the expression of ER chaperones, folding enzymes, and ERAD components in response to ERS [37,38]. The unspliced XBP1 mRNA (XBP1u) translates into highly unstable proteins [15]. RIDD can cause the degradation of certain mRNAs, thereby reducing the proteins that enter the ER [39,40].

PERK is a type I ER membrane protein, and its ER luminal domain is similar in sequence and structure to that of IRE1 LD [41]. When the cytoplasmic kinase structural domain of PERK is activated via trans-autophosphorylation, it inactivates eukaryotic initiation factor 2α (eIF2α). On one hand, eIF2α phosphorylation completely inhibits protein translation, and on the other hand, it facilitates the translation of a specific set of mRNAs [26]. This implies that the activation of PERK can reduce the number of proteins entering the ER as an adaptive response to ERS and, additionally, promote the production of proteins adapted to ERS [42]. eIF2α phosphorylation selectively increases the translation of activating transcription factor 4 (ATF4), which contributes to the expression of genes regulating protein folding, resistance to oxidative stress, and amino acid metabolism, and reduces the accumulation of misfolded proteins in the ER [43]. Under prolonged ERS, ATF4 induces the expression of transcription factor C/EBP homologous protein (CHOP, also known as DDIT3) and upregulates the expression of pro-apoptotic BCL2-related proteins to promote cell death [14].

ATF6 is a type II transmembrane protein with two isoforms, ATF6α and AFT6β, similar to IRE1. AFT6α is primarily associated with ERS, and AFT6β primarily plays a regulatory role [36]. Under ERS, ATF6 is transported to the Golgi apparatus, where it is proteolytically hydrolyzed by site 1 and site 2 proteases (S1P and S2P, respectively), and cleaved ATF6 enters the nucleus to regulate the expression of genes encoding the components of XBP1, BiP, and ERAD [44,45,46].

It has been shown that the IRE1α pathway is associated with apoptosis, differentiation, metastasis, and drug resistance in cancer cells [47,48]. In osteosarcoma, the knockdown of XBP1 leads to tumor growth inhibition [49]. P97, also recognized as valosin-containing protein (VCP) and cytokinesis cyclin 48 (CDC48), belongs to the ATPases associated with multiple cellular activities (AAA+) family [50]. It can affect proteostasis and functions as an important regulator of ERAD [51,52]. The inhibition of P97 in osteosarcoma cells was found to inhibit the ERAD pathway, allowing the accumulation of unfolded proteins in cells, and subsequently activate PERK- and IRE1-related UPR pathways, namely the IRE1α-XBP1s-CHOP pathway and the PERK-eIF2α-ATF4-CHOP pathway, to promote protein degradation and apoptosis [53].

Studies have shown that ATF6α activation is an indicator of poor prognosis in osteosarcoma and improves cell survival after chemotherapy [54]. Polo-like kinase 4 (PLK4) belongs to the serine/threonine protein kinase family, and its deficiency inhibits cell proliferation in U2OS osteosarcoma [55]. Under ERS, ATF6 is activated and binds to the PLK4 promoter to recruit C/EBPβ, thereby inhibiting apoptosis in osteosarcoma cells [56]. It was found that upregulation of only pro-apoptotic BH3 family members Noxa and Puma and downregulation of transcription factor E2F1 contributed to ERS-induced cell death under long-term ERS. In osteosarcoma, upregulation of ATF6 and E2F7 during sustained ERS suppressed E2F1 expression, upregulation of E2F7 was dependent on activation of XBP1, and downregulation of E2F1 enhanced Noxa and Puma expression and promoted ER stress-induced apoptosis [57].

Both GPR78 and GPR94 are markers of ERS [58]. GRP94 and GRP78 are ER chaperone proteins. Under non-stress conditions, GRP94 and GRP78 maintain ER protein folding environment and protein balance by binding to IRE1, ATF6, and PERK [59]. Under ER stress, GRP78 and GRP94 dissociate from IRE1, ATF6, and PERK, and then bind to unfolded and misfolded proteins to activate UPR. Free IRE1, ATF6, and PERK trigger downstream signal transduction, which increases ER protein folding ability and ER-related protein degradation through transcription of various chaperone-related genes, namely GRP78 and GRP94. Once UPR fails to control the level of unfolded and misfolded proteins, ER activates the apoptotic signal and activates the death factor CHOP to induce apoptosis and cell death [58]. GPR78 and GPR94 are associated with chemotherapy resistance in osteosarcoma. Studies have shown that GRP78 expression is enhanced in osteosarcoma tissues of patients resistant to both doxorubicin and platinum-based drugs, suggesting that GRP78 is involved in ERS-induced drug resistance in osteosarcoma cells. This mechanism of drug resistance may be an adaptive mechanism of cancer cells in response to ERS. By activating UPR, cancer cells upregulate proteins such as GPR78 and GPR94, thus promoting ubiquitination degradation of CHOP protein, reducing the activity of CHOP, and restoring the folding environment and protein balance of ER protein, thus enabling cancer cells to survive [60]. In addition, normal osteoblasts expressed ATF4, which significantly inhibited the occurrence of OS. RET is a receptor tyrosine kinase (RTK) required for normal cell development. The absence of RET upregulates ATF4, which accelerates the turnover of RET proteasomes by over-recruitment of its trans-activated E3 ligase CPL-C, thereby enhancing the chemotherapy effect of BTZ and preventing BTZ resistance in osteosarcoma cells. However, the combination of GPR78 and RET interferes with the interaction between RET and ATF4, down-regulating ATF4 and inhibiting the anti-tumor effect of BTZ, thus leading to chemotherapy resistance [61]. In osteosarcoma, GPR94 does not affect tumor proliferation or migration, but it decreases tumor sensitivity to chemotherapy [62].

The ISR was first identified in 2002 [63,64] and is an adaptive signaling pathway in eukaryotic cells that is activated by a variety of pathological stimuli, including hypoxia, amino acid deprivation, glucose deprivation, and viral infection [65], and the ERS can also activate the ISR [66]. The ISR is mediated by four kinases: PERK, double-stranded RNA-dependent protein kinase (PKR), heme-regulated eIF2α kinase (HRI), and general control non-derepressible 2 (GCN2). Each of these four kinases senses different stimuli, PERK is activated when unfolded or misfolded proteins accumulate in the ER, PKR is activated by binding to double-stranded RNA (dsRNA), HRI is sensitive to heme deficiency, and GCN2 is induced by amino acid deficiency, and they all exert their effects by causing phosphorylation of eIF2α [67]. Under short-term stress, ISR exerts a pro-cell survival effect and restores cellular homeostasis, whereas, under sustained and severe stress, ISR induces apoptosis [68]. PERK-eIF2α-ATF4-CHOP is a common pathway between UPR and ISR, which in osteosarcoma crosstalk with autophagy, oxidative stress, and affects the development of osteosarcoma, as will be described below.

Autophagy is a conserved pathway active in all eukaryotes and it can be triggered by a variety of stress factors, such as nutritional deficiency, hypoxia, oxidative stress, and chemical drugs, and it plays a dual role in cancer cells. On the one hand, it can eliminate oncogenic protein substrates, damaged organelles, and inhibit tumor growth [69]; on the other hand, it can provide nutrition and energy to tumors in a state of hypoxia and nutritional deficiency, and promote tumor cell survival [70] and metastasis [71]. ERS and autophagy were found to be associated with post-chemotherapeutic drug resistance in osteosarcoma, in which PERK plays a key role [72,73,74]. Target of rapamycin (TOR) is a serine/threonine protein kinase that acts as a critical modulator of autophagy, and activation of the mammalian TOR (mTOR) pathway inhibits autophagy [75]. Sestrins (SESN) can regulate autophagy [76]. Recent studies have shown that the cellular expression of Sestrin2, which belongs to the SESN family, increases during chemotherapy for osteosarcoma, which in turn inhibits PERK-eIF2α-CHOP activation, leading to a decrease in CHOP-induced apoptosis. In addition, low CHOP expression results in a decrease in p-mTOR protein levels, and autophagy is subsequently activated, which protects cells by reducing apoptosis [73]. Oh et al. showed that ERS-activated autophagy is an important contributor to radiosensitivity in osteosarcoma [77]. Their study showed that high linear energy transfer (LET) radiation can induce autophagy and promote apoptosis through two ERS pathways, contributing to cellular sensitivity to high levels of LET radiation. Under ERS, PERK is activated, phosphorylates eIF2α, and mediates autophagy through the ATF4-CHOP-Akt-mTOR axis. Moreover, IRE1 is activated, binds to TNFR-associated factor 2 (TRAF2) to form the IRE1-TRAF2-ASK1 complex, and then phosphorylates JNK, thus allowing Bcl-2 to dissociate from Beclin 1 to mediate autophagy.

Oxygen-demanding cells produce ROS during metabolism, and excessive ROS can damage DNA and induce apoptosis [78]. ERS was found to be closely associated with oxidative stress in various diseases [79]. The increase in ROS affects endoplasmic reticulum homeostasis and induces ERS [79,80]. Miao et al. showed that oxidative stress induced by the use of bortezomib in combination with adriamycin for osteosarcoma activated the PERK/eIF2α/ATF4/CHOP axis, thereby inducing apoptosis [81]. Huang et al. demonstrated that drug-laden nanoparticles promoted the generation of ROS in osteosarcoma cells and participated in ERS-induced apoptosis through the JNK/p53/p21 pathway [82]. Unfolded proteins in the ER also activate ROS; the production of approximately a quarter of the ROS content in cells may be related to the formation of disulfide bonds in the ER during oxidative protein folding [83]. One of the mechanisms proposed for the formation of disulfide bonds in the ER that generates ROS is that ER oxidoreductin 1 (ERO1) synergizes with reduced protein disulfide isomerase (PDI) to produce ROS [79]. ERO1 is a producer of H₂O₂ in the intracellular UPR pathway and is regulated by CHOP [84,85]. CYT997 (lexibulin) (a microtubule-targeting agent), when used to treat osteosarcoma, damages mitochondria to produce ROS and also induces ERS, which exacerbates oxidative stress through the PERK/ eIF2α/CHOP/ERO1 axis, while ROS produced by the mitochondrial pathway also exacerbates ERS, ERS and oxidative stress enhance each other to promote apoptosis [72].

PI3K/Akt is a common and important signaling pathway in cells, which is related to phosphatidylinositol and is also an RTK-mediated derived signaling pathway. AKT is the core of this pathway and numerous molecules and pathways are simultaneously connected downstream, such as VEGF and FOXO [86]. The PI3K/Akt pathway is a key regulator activated during cellular stress [87]. The activation of mTOR can lead to the increased synthesis of various proteins [88]. Tumor cells can escape drug-induced growth inhibition and death through the PI3K/Akt/mTOR pathway [89], whereas the PI3K/Akt/mTOR pathway activates the autophagic pathway, which in turn protects tumor cells [90]. Studies have shown that the inhibition of the PI3K/Akt/mTOR pathway exacerbates ERS in disease cells [91,92]. In addition, NF-κB transcription factors are important regulators of various responses, such as stress response, apoptosis, and differentiation, and are closely linked to other pathways [93], which is more important for the survival and drug resistance of osteosarcoma [94]. The PI3K/Akt/NF-κB signaling pathway is associated with metastasis and invasion in osteosarcoma [95]. Yan M et al. found that when osteosarcoma was treated with cisplatin, the UPR was activated, and both branches of PERK and IRE1 activated NF-κB, thus preventing the cells from acquiring chemoresistance to cisplatin [96].

The Wnt-3a/β-catenin pathway is associated with tumorigenesis and progression [99,100,101]. Some studies have shown that this pathway is associated with ERS [102,103]. However, the specific role of the Wnt-3a/β-catenin pathway in osteosarcoma is still unclear. Several studies have shown that this pathway participates in the regulation of proliferation and apoptosis of osteosarcoma cells; e.g., zinc inhibits the proliferation and promotes the apoptosis of osteosarcoma cells via the activation of the Wnt-3a/β-catenin signaling pathway [104]. MicroRNA-152 suppresses the growth of osteosarcoma cells via the Wnt/β-catenin signaling pathway [105]. However, Yang et al. showed that α-mangostin induced ERS by promoting ROS production, which in turn inhibited the Wnt/β-catenin signaling pathway and activated the caspase-3/8 cascade, causing apoptosis in osteosarcoma cells [106]. At the same time, Jiang et al. demonstrated that histone methyltransferase SETD2 can inhibit the growth of osteosarcoma cells by inhibiting Wnt-3a/β-catenin signaling pathway [107].

Several microRNAs have been reported to be significantly associated with ERS triggered in osteosarcoma cells, notably miR-1281 and miR-663a [108,109,110]. Recent studies have shown that miR-1281 and miR-663a exert a significant effect on ERS triggered in osteosarcoma cells [111,112]. P53 is a tumor suppressor. USP39 (Ubiquitin precific peptidase 39) plays an important role in osteosarcoma. USP39 knockdown inhibits cell growth and enhances cell apoptosis. In osteosarcoma cells under ER stress, P53 directly binds to the promoter of miR-1281, and down-regulates USP39 in osteosarcoma cells through the p5-Mir-1281-USP39 axis, an ERS response pathway, to inhibit and promote cell apoptosis [111]. ZBTB7A is a member of the POK transcriptional repressor family, which is expressed aberrantly in some cancers and plays an important role in tumorigenesis, including that in osteosarcoma [113,114,115]. ZBTB7A is a target gene of miR-663a. ERS induces miR-663a and protects osteosarcoma from ERS-induced apoptosis by inhibiting the expression of lncRNAGAS5. This change is achieved by Mir-663a-ZBTB7A-lncRNAGAS5 [112].