Akt signaling in osteosarcoma.

CCA-13873; No of Pages 11 Clinica Chimica Acta xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Clinica Chimica Acta

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Invited critical review

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PI3K/Akt signaling in osteosarcoma

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Jian Zhang a, Xiao-Hua Yu b, Yi-Guo Yan a, Cheng Wang a, Wen-Jun Wang a,⁎

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Article history: Received 5 October 2014 Received in revised form 24 November 2014 Accepted 9 December 2014 Available online xxxx

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Keywords: OS PI3K Akt PTEN mTOR

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Osteosarcoma (OS) is the most common nonhematologic bone malignancy in children and adolescents. Despite the advances of adjuvant chemotherapy and significant improvement of survival, the prognosis remains generally poor. As such, the search for more effective anti-OS agents is urgent. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is thought to be one of the most important oncogenic pathways in human cancer. An increasing body of evidence has shown that this pathway is frequently hyperactivated in OS and contributes to disease initiation and development, including tumorigenesis, proliferation, invasion, cell cycle progression, inhibition of apoptosis, angiogenesis, metastasis and chemoresistance. Inhibition of this pathway through small molecule compounds represents an attractive potential therapeutic approach for OS. The aim of this review is to summarize the roles of the PI3K/Akt pathway in the development and progression of OS, and to highlight the therapeutic potential of targeting this signaling pathway. Knowledge obtained from the application of these compounds will help in further understanding the pathogenesis of OS and designing subsequent treatment strategies. © 2015 Published by Elsevier B.V.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview of the PI3K/Akt signaling pathway . . . . . . . . . . . . . . 2.1. Activation of the PI3K/Akt signaling pathway . . . . . . . . . . 2.2. PTEN as a main negative regulator of the PI3K/Akt signaling pathway Roles of the PI3K/Akt pathway in OS pathogenesis . . . . . . . . . . . 3.1. Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Proliferation and invasion . . . . . . . . . . . . . . . . . . . 3.3. Cell cycle progression . . . . . . . . . . . . . . . . . . . . . 3.4. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Chemoresistance . . . . . . . . . . . . . . . . . . . . . . . Treatment targeting the PI3K/Akt pathway in OS . . . . . . . . . . . . 4.1. PI3K inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Dual PI3K/mTOR inhibitors . . . . . . . . . . . . . . . . . . 4.3. Akt inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 4.4. mTOR inhibitors . . . . . . . . . . . . . . . . . . . . . . . 4.5. PTEN activators . . . . . . . . . . . . . . . . . . . . . . . 4.6. Natural compounds from plants . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . .

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Department of Spine Surgery, The First Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China Life Science Research Center, University of South China, Hengyang, Hunan 421001, China

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Abbreviations: OS, osteosarcoma; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; mTOR, mammalian target of rapamycin; RTKs, receptor tyrosine kinases; PDGFR, platelet-derived growth factor receptor; IGF1R, insulin-like growth factor 1 receptor; PIP2, phosphatidylinositol 4,5-biphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PDK1, phosphoinositide-dependent kinase 1; PH, pleckstrin homology; CDK, cyclin-dependent kinase; NF-κB, nuclear factor-κB; MDM2, Murine double minute 2; Rb, retinoblastoma; CSCs, cancer stem cells; CXCR7, CXC chemokine receptor 7; GA, gallic acid; miRNAs, microRNAs; TNF, tumor necrosis factor; HIF-1, hypoxia-inducible factor-1; ET-1, endothelin-1; ETAR, endothelin A receptor; MMP, matrix metalloproteinase. ⁎ Corresponding author. Tel./fax: +86 734 8578579. E-mail address: [email protected] (W.-J. Wang).

http://dx.doi.org/10.1016/j.cca.2014.12.041 0009-8981/© 2015 Published by Elsevier B.V.

Please cite this article as: Zhang J, et al, PI3K/Akt signaling in osteosarcoma, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2014.12.041

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Disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.1. Activation of the PI3K/Akt signaling pathway

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PI3Ks constitute a lipid kinase family and include three classes (I–III) according to their substrate preferences and sequence homology. Among these, class I PI3Ks are the most relevant to cancers, which are further divided into two subclasses: class IA (PI3Kα, PI3Kβ and PI3Kδ) and class IB (PI3Kγ). These class I PI3Ks form a heterodimer that is made up of a catalytic subunit and a regulatory subunit. The catalytic subunits contain p110α, β, γ and δ, which are encoded by the genes PIK3CA, PIK3CB, PIK3CG, and PIK3CD, respectively [6]. The regulatory subunits comprise p85α (p85α, p55α, p50α), p85β and p55γ, which are encoded by the genes PIK3R1, PIK3R2, and PIK3R3, respectively. In vitro, each p110 subunit has the ability for binding to any of the regulatory subunits. PI3Kα, PI3Kβ and PI3Kδ are activated by receptor tyrosine

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The most important downstream effector of Akt signaling is mTOR, a 289 kDa serine/threonine kinase. In the mammalian cells, it exists in two multiprotein complexes, namely mTORC1 and mTORC2. mTORC1 plays a critical role in the carcinogenesis of many cancers including OS, and it is composed of a regulatory-associated protein of mTOR (Raptor), mTOR, mammalian LST8/G-protein β-subunit like protein (GβL), proline-rich Akt substrate 40 (PRAS40), and DEP domain containing mTOR-interacting protein (Deptor) [11]. However, mTORC2 consists of Rictor, mTOR, GβL, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1). Akt can phosphorylate tuberous sclerosis complex (Tsc) 2, a tumor suppressor, leading to inhibition of the Tsc1/Tsc2 heterodimers. The inhibited Tsc1/Tsc2 heterodimers allow GTP-binding protein Rheb (Ras homolog enriched in brain) to remain in its active GTP-bound state, causing an increase in mTORC1 activity [12]. Activated mTORC1 mediates the phosphorylation of ribosomal protein S6 kinases (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), the latter of which causes the release of eukaryotic translation initiation factor 4E (eIF4E). These directly lead to protein translation and cell cycle progression. In addition, mTORC2 acts as an activator of Akt, thus providing a positive feedback loop between Akt and mTOR to further facilitate cell proliferation and survival [13].

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In addition to mTOR, Akt can phosphorylate and deactivate glycogen synthase kinase 3β (GSK3β), a serine/threonine kinase, resulting in increased cyclin D1 and Myc [14]. Myc is an oncoprotein that upregulates cyclin-dependent kinase (CDK) 4. The Akt-mediated inhibition of forkhead box O1 (FOXO1) stimulates the downregulation of the CDK inhibitors p27 and p21, which significantly accelerates cell cycle [15]. Another target activated by Akt is nuclear factor-κB (NF-κB), a central signaling factor involved in the development and progression of human cancers as well as in the acquisition of drug-resistant phenotype in highly aggressive malignancies [6]. Akt increases the activity of inhibitor of κB (IκB) kinase (IKK), which leads to phosphorylation and degradation of IκB and subsequent release of NF-κB. Once NF-κB becomes free, it can rapidly translocate to the nucleus and trigger the transcription of many target genes. Akt also plays an important role in suppressing cellular apoptosis. On one hand, it is able to diminish pro-

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Once activated, the catalytic subunit of PI3Ks converts the substrate phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3) [8]. PIP3 then recruits a subset of signaling proteins with pleckstrin homology (PH) domains to plasma membrane, including phosphoinositide-dependent kinase 1 (PDK1) and Akt, also known as protein kinase B (PKB). Akt contains three isoforms (Akt1, Akt2, and Akt3) which have high homology at the amino acid level and are activated through two key phosphorylation events. Akt is partially activated through an initial phosphorylation at threonine 308 (Thr308) in the kinase domain by PDK-1, and then fully activated by the subsequent phosphorylation at serine 473 (Ser473) in the Cterminal regulatory domain by several potential kinases such as PDK1, integrin-linked kinase (ILK), DNA-dependent protein kinase (DNAPK), mTORC2 or Akt itself [9,10]. Schematic structure of AKT protein is presented in Fig. 1A. Thereafter, activated AKT translocates from cell membrane to the cytoplasm and nucleus, where it can phosphorylate, activate, or suppress many downstream targets to regulate various cellular functions.

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Osteosarcoma (OS) is the most common primary bone sarcoma that occurs predominantly in children and adolescents, comprising approximately 20% of all bone tumors and about 5% of pediatric tumors overall. Although the malignancy can form in any bone, the origin is most frequent in the metaphyseal regions of the distal femur, proximal tibia and proximal humerus, with a male predominance. OS is characterized by a local invasion of bone and soft tissues, loss of the affected extremity's functions and distant metastasis, most often to the lung. In patients without metastases at the time of diagnosis, combination of aggressive surgical resection and chemotherapy has led to a significant improvement of five-year survival rate (60–70%) [1]. However, these conventional therapeutic approaches have reached a survival plateau, and chemotherapy can cause drug-resistance and produce a variety of side effects, such as cardiotoxicity, nephrotoxicity, hearing loss and risk of local relapse. In contrast, cases with metastasis or recurrence have a b20% chance of long-term survival despite the use of chemotherapeutic drugs [2]. Thus, there is an urgent need for the development of more effective anti-OS agents with enhanced ability to remove systemic tumor burden and decrease toxicity in healthy tissues. Despite the fact that the etiology of OS is not fully elucidated, there has been a large body of evidence that the disease is involved in dysregulation of multiple intracellular signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This pathway is negatively regulated by phosphatase and tensin homolog (PTEN) and can activate many downstream targets such as mammalian target of rapamycin (mTOR). The PI3K/Akt pathway is a cascade of events, which plays a critical role in the broad variety of physiological and pathological processes. It is well established that the pathway is one of the most important oncogenic pathways in almost all kinds of human cancers [3]. In fact, amplifications and mutations have been identified in nearly every member of the pathway [4]. In OS, the PI3K/AKT signaling pathway is deregulated in the majority of localized disease and 100% of advanced-stage disease, which implies that alterations in this pathway may be a pre-requisite for OS progression [5]. Recently, many small molecule compounds targeting the PI3K/AKT signaling pathway have been developed, and show promise for improving survival of OS patients. In this review, we briefly summarize activation and regulation of the PI3K/AKT signaling pathway, and then focus on recent progress regarding the pathogenetic significance and targeted therapy of this complex pathway in OS.

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kinases (RTKs) such as platelet-derived growth factor (PDGF) receptor (PDGFR), insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor (EGF) receptor (EGFR); however, PI3Kγ is activated by G-protein-coupled receptors (GPCRs) and small GTPase Ras [7].

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PTEN was firstly identified in 1997 as the relevant gene in a region of chromosome 10 that is often lost in late-stage human tumors. As a dualspecificity phosphatase, PTEN has protein phosphatase activity and lipid phosphatase activity. The human PTEN gene maps to chromosome 10q23.3, and consists of nine exons. It encodes a 5.5 kb mRNA that

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apoptotic Bad and Bax levels but elevate anti-apoptotic B-cell lymphoma 2 (Bcl-2), Bcl-xl and myeloid cell leukemia 1(Mcl1) [16]. On the other hand, it upregulates the expression of Murine double minute 2 (MDM2) and then reduces the release of p53, which can induce apoptosis in the presence of DNA damage or oncogenic stress [17]. The overall effects of PI3K/Akt signaling pathway are to enhance cellular replication and survival and to reduce growth prevention and apoptosis (Fig. 2).

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Fig. 1. Schematic of Akt and PTEN protein structures.

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Fig. 2. Illustration of the PI3K/Akt signaling pathway. After activation, PI3K converts PIP2 into PIP3, but PTEN can reverse this process. PIP3 then phosphorylates and activates Akt in a PDK1dependent manner, and mTORC2 also contributes to Akt activation. Activated Akt increases Rheb-GTP levels by inhibiting the TSC1/TSC1 heterodimer formation, leading to activation of mTORC1. mTORC1 subsequently mediates the phosphorylation of S6K and 4E-BP1, and the latter promotes the release of eIF4E. These result in increased protein synthesis and cell growth. Elevation of IKK activity induced by Akt facilitates IκB degradation, which causes the liberation and nuclear entry of NF-κB for gene transcription. Akt negatively modulates GSK3β and FOXO1. Reduced GSK3β augments the expression of cyclin D1 and Myc, the latter of which leads to a significant increase of CDK4 levels. Meanwhile, suppression of FOXO1 decreases p27 and p21 contents and in turn enhances CDK expression. Consequently, upregulation of these molecules significantly promotes cell cycle progression. In addition, Akt inhibits apoptosis through upregulation of Bcl-xl, Bcl-2 and Mcl1 as well as downregulation of Bad, Bax and p53.


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Considering the important role of the PI3K/AKT pathway in normal cell physiology, it is not surprising that the pathway is highly deregulated in many types of cancers. Kinome profiling has identified active Akt signaling in the majority of OS cell lines [20]. Recently, a large body of evidence suggests that dysregulation of this pathway has been involved in multiple pathological processes of OS, including tumorigenesis, proliferation, invasion, cell cycle progression, apoptosis, angiogenesis, metastasis and chemoresistance (Fig. 3).

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3.1. Tumorigenesis

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Despite OS occurrence is correlated with several genetic predisposition conditions, the majority of OS is sporadic without familial patterns. Our current understanding of OS etiology is rather limited. Nevertheless, there is growing evidence that the disease is closely associated with the activation of many oncogenes, such as c-Myc, MDM2 and cyclin D1 [21]. The c-Myc product is involved in regulating cell growth and DNA replication. c-Myc amplification has been found in 7–12% of OS [22]. More recently, Zhai et al. reported that phosphorylated Akt markedly upregulated c-Myc expression along with a significant

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increase in OS cell proliferation and invasion [23]. MDM2, located in chromosome 12q13, encodes a protein that can negative regulate p53 levels by binding to p53 for degradation. As mentioned above, activated PI3K/Akt pathway can strongly induce MDM2 transcription. It is possible that this pathway contributes to OS genesis through the upregulation of MDM2. Nevertheless, further studies are necessary to test the hypothesis. In addition, cyclin D1 plays a key role in G1 to S phase transition and acts as a downstream target of mTORC1. High levels of cyclin D1 have been detected in 22% of OS, and its amplification has been reported in 4% of OS [24]. Several studies have shown that activation of the PI3K/Akt pathway is able to increase cyclin D1 expression and then inhibit OS cell apoptosis and promote cell growth [25,26]. Thus, these data suggest that activation of c-Myc, MDM2 and cyclin D1 mediated by the PI3K/Akt pathway may play an important role in OS tumorigenesis. Genes involving DNA repair and tumor suppressor pathways help maintain the integrity of critical cellular processes. Defects in these genes often lead to carcinogenesis [27]. The tumor suppressor gene TP53 is located in chromosome 17p13.1, a region frequently identified as abnormal in OS [28]. Its alterations observed in OS contain point mutations (20%–30%, mostly missense mutations), gross gene rearrangements (10%–20%), and allelic loss (75%–80%) [29]. The association of TP53 with OS is further supported by Li-Fraumeni syndrome, an autosomal dominant disorder which is characterized by a germline mutation of TP53 and can develop into OS [30]. The product encoded by TP53 is p53, which is capable of mediating tumor prevention through cell cycle arrest and induction of apoptosis after DNA injury or oncogene activation. In cultured human OS cells, blockade of the Akt/mTORC1 signaling has been shown to stimulate p53 release, which is responsible for a marked increase of cell apoptosis and growth inhibition [31]. Conversely, canine OS cell lines (CO2, CO3, CO5 and CO7) with PTEN inactivation constitutively expressed high levels of the phosphorylated form of Akt, leading to reduced p53 [32]. Retinoblastoma (Rb) is also a tumor suppressor. Genetic alterations in Rb have been found in up to 70% of sporadic OS patients. Homozygous deletions of Rb are seen in

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specifies an open reading frame (ORF) of 403 amino acids. The translation product, a 53 kDa protein with homology to tensin and protein tyrosine phosphatases (PTPs), contains an N-terminal phosphatase domain, a C-terminal C2 domain, a PDZ (PSD-95, DLG1, and ZO-1)-binding motif, and two PEST (proline, glutamic acid, serine, and threonine) sequences involved in protein degradation (Fig. 1B) [18]. The CX5R(S/T) motif is essential for PTEN lipid phosphatase activity, and it locates within an active site surrounding the catalytic signature with three basic residues. The structure endows PTEN with its preference for acidic phospholipid substrates, especially PIP3, converting PIP3 back to PIP2 (Fig. 2) [19]. Thus, loss of PTEN activity leads to permanent activation of the PI3K/Akt pathway.

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Fig. 3. Activation of the PI3K/Akt pathway promotes multiple pathological processes of OS initiation and development.


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Dysregulation of cell cycle is intimately correlated with aggressive biological behavior of malignant tumors and thus functions as a fundamental hallmark of cancer progression. Cell cycle is predominantly modulated by cyclins and CDKs. The cyclin family members interact with CDKs to form the active heterodimeric complexes that are required to pass through specific phases of cell cycle [54]. Interestingly, the PI3K/ Akt signaling pathway is considered to be a positive regulator of cell cycle in OS cells by enhancing the expression of cyclins and CDKs. Synovial sarcoma X breakpoint (SSX) is a transcription factor with elusive oncogenic functions expressed in many human tumors of epithelial and mesenchymal origin. In OS cell lines, overexpression of SSX strongly promoted cell cycle progression due to upregulation of Akt and downstream cyclin A [55]. Conversely, the knockdown of high mobility group nucleosome binding domain 5 (HMGN5), a member of the high mobility group protein family, significantly inhibited the activities of PI3K and Akt and then decreased cyclin B1 expression, leading to the induction of cell cycle arrest in U2OS and SaO2 cells [56]. Carotenoids such as fucoxanthin and its metabolite fucoxanthinol are natural pigments and exhibit a variety of biological functions. Exposure of OS cells to fucoxanthinol caused cell cycle arrest at G1 phase in part by inhibiting Akt activation and subsequent expression of CDK4, CDK6 and cyclin E [57]. Blockade of the PI3K/Akt pathway by metastasis-associated in colon cancer-1 (MACC1) downregulation also prevented G0 to G1 phase transition in U2OS cells, which was attributed to a significant decrease in CDK4 and CDK6 levels [58]. In addition to cyclins and CDKs, the PI3K/Akt signaling pathway has a significant effect on other factors involving cell cycle control. It is well established that CDKs are inhibited by two families of inhibitors [59]. The cyclin inhibitor protein/kinase inhibitor protein (CIP/KIP) family includes p21CIFI/WIFI, p27KIP1 and p57KIP2, which can inhibit any cyclin/CDK complex. The inhibitor of CDK4 (INK4) group consists of p15INK4b, p16 INK4a, p18 INK4c and p19 INK4d, which specifically suppress CDK4

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Proliferation and invasion play a crucial role in the progression of malignant tumors, including OS. There is accumulating evidence that the PI3K/Akt pathway promotes these aggressive behaviors. Wnt5a is classified as a member of non-transforming Wnt family and plays complicated roles in oncogenesis. Overexpression of Wnt5a has been shown to stimulate the migration of MG-63 via enhancing phosphorylation of PI3K and Akt [38]. The interaction between tumor cells with platelets contributes to the progression of tumor malignancy. Co-culture of platelets with MG-63 or HOS cells, could induce platelet aggregation and enhance the proliferation of each cell line in vitro through activation of the PDGFR/Akt signaling axis [39]. microRNAs (miRNAs) are a class of endogenously expressed, small noncoding RNAs, which can negatively modulate gene expression by targeting mRNAs for translational repression and/or cleavage [40]. Silencing miR-223 increased heat shock protein (HSP) 90B1 expression and subsequent protein levels of PI3k, p-Akt and mTOR, which promoted MG-63 cell proliferation [41]. PTEN, known as a potent negative regulator of Akt, is thought to play a critical role in controlling the PI3K/Akt signaling activation [42]. Any alteration in the PTEN expression may also contribute to the progression of OS. Indeed, ectopic expression of miR-221 directly targeted PTEN and then activated the PI3K/Akt signaling pathway, leading to more OS cell survival [43]. Overexpression of miR-17 substantially promoted OS cell proliferation, migration and invasion by directly binding to 3′-untranslated region (UTR) of PTEN [44]. In addition, PTEN has been identified as a target of miR-128, and upregulation of miR-128 significantly increased while its suppression by its antisense reduced the proliferation of MG-63 and U2OS cells in a PTEN-dependent manner [45]. In agreement, downstream Akt signaling was activated or inhibited by miR-128 overexpression or knockdown, respectively [45]. Thus, these findings suggest that miR-17, miR-128 and miR-221 can induce the malignant phenotype of OS through blockade of PTEN-mediated inhibition of PI3K/Akt signaling pathway, and in vivo delivery of these miRNA inhibitors may have a potential therapeutic benefit for OS patients. On the other hand, blockade of the PI3K/AKT signaling cascade protects against OS cell proliferation, adhesion and invasion. A recent experimental study has revealed that a knockdown of CXC chemokine receptor 7 (CXCR7) inhibits proliferative activity and invasive potential of OS cell lines MG-63 and U2OS partially through decreased expression of PI3K and AKT [46]. In human OS cell lines Saos-2, MG-63 and SJSA-1, treatment with selective endothelin A receptor (ETAR) antagonist BQ123 attenuated the activities of PI3K and AKT and then suppressed cell invasion and viability, and these effects were reversed by the PI3K inhibitor BKM120 [47]. Kuijjer and colleagues analyzed genome-wide

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gene expression data of OS cell lines U2OS and HOS [20]. They found that Akt expression was upregulated, and addition of the allosteric Akt inhibitor MK-2206 resulted in inhibition of cell proliferation [20]. Downregulation of fatty acid synthase (FAS) significantly decreased the expression levels of phosphorylated PI3K (p-PI3K) and Akt (p-Akt), and subsequently reduced the proliferative and invasive abilities of U2OS cells [48,49]. Gallic acid (GA) is known to possess multiple pharmacological activities including antitumor effects. Administration of GA has been shown to block U2OS cell migration and invasion through downregulation of the PI3K/Akt signaling pathway, indicating a protective role for GA in controlling the aggressive biological behavior of OS [50]. Matrix metalloproteinases (MMPs) are responsible for the degradation of collagens within extracellular matrix (ECM) and thus play a critical role in promoting tumor cell invasion. Recently, Liu et al. have reported that the knowndown of iroquois homeobox 2 (IRX2) with a lentivirus vector can diminish the proliferative and invasive capacities of OS cells via downregulation of the Akt/MMP-9 signaling [51]. Aminopeptidase N (APN), a Zn+2-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion mechanism. Addition of APN inhibitor bestatin to human OS cells markedly prevented the activation of the PI3K/Akt pathway and then reduced the activities of MMP-2 and MMP-9, leading to decreased cell migration and invasion, whereas a converse effect was observed in cells treated with interleukin 6 (IL-6), a stimulator of APN [52]. Deguelin, a naturally occurring rotenoid, is known to be an Akt inhibitor and to exhibit cytotoxic effects. Administration of Deguelin was also able to attenuate MMP-2 and MMP-9 secretion and thereby suppress the migration and invasion of U2OS cells due to Akt inactivation [53]. Thus, these studies provide sufficient evidence supporting the involvement of MMP-2 and MMP-9 in PI3K/Akt-mediated OS cell invasion.

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23% of tumors, whereas point mutations appear in 6% of tumors [1]. Rb functions as an important regulator of G1/S cell cycle progression. During G1/S transition, Rb becomes phosphorylated and inactive, resulting in the activation of E2F transcription factors that bind to the phosphorylated Rb protein and facilitate DNA synthesis and G1 to S transition [33]. It is suggested that enhanced Akt activity dramatically promotes Rb phosphorylation and OS cell proliferation [34]. Overall, the PI3K/ Akt-induced inactivation of p53 and Rb exerts a positive effect in the etiology of OS. Cancer stem cells (CSCs) have the capacity to self-renew and differentiate, but the mechanisms that strictly regulate these processes under normal conditions are deregulated, leading to their expansion and production of aberrantly differentiated progeny [35]. These cells are considered to play a critical role in tumorigenesis of many malignancies, including OS [36]. The PI3K/Akt pathway is known to be involved in regulation of tumor cell fates, such as proliferation, cell cycling, survival and apoptosis. It has been reported that PI3K inhibitor markedly reduced PI3K activity and subsequently induced G0/G1 cell cycle arrest and apoptosis in OS CSCs [37]. Thus, survival of CSCs mediated by activated PI3K/Akt pathway may also contribute to OS initiation.

E

253 254

5


319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347

350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380

411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444

C

409 410

E

407 408

R

405 406

R

403 404

O

401 402

C

399 400

N

392 393

U

390 391

F

Apoptosis, also termed programmed cell death, plays a crucial role in controlling embryonic development and maintaining tissue homeostasis of multicellular organisms. In mammalian cells, it is initiated through two well-characterized pathways. One is extrinsic pathway, mediated by dead receptors, including tumor necrosis factor (TNF) receptor, TNF-related apoptosis-inducing ligand (TRAIL) receptor and Fas. The other is intrinsic pathway, mediated by molecules released from the mitochondria, including cytochrome c, apoptotic protease-activating factor 1 (Apaf-1). Both pathways cause the activation of the initiator caspases, which then activate effector caspases to eliminate damaged cells. Dysregulation of apoptosis has been involved in the pathology of a number of diseases, including tumor genesis and development. Recently, the role of the PI3K/Akt pathway in OS cell apoptosis has obtained a great attention. Upregulation of HSP70 expression was found to activate the PI3K/Akt signaling and then decrease caspase-3 activity, which partially prevented human OS cells from undergoing apoptosis [63]. On the other hand, knockdown of Akt2 increased the expression of p53 upregulated modulator of apoptosis (PUMA), leading to sensitivity of Saos-2 cells to apoptosis induced by cisplatin treatment [64]. Lapatinib, an inhibitor of human EGFR2 phosphorylation, could enhance apoptosis of U2OS and MG-63 cells via downregulation of the EGFR2/PI3K/Akt pathway [65]. As an ECM remodeling enzyme, lysyl oxidase (LOX) is involved in the development and progression of many types of tumors. Xu and colleagues have demonstrated that overexpression of LOX significantly reduces the activities of PI3K and Akt, with concomitant increase in OS cell apoptosis [66]. U2OS cells treated with the lyophilized red wine exhibited a significant elevation of apoptotic rate through blockade of the PI3K/Akt signaling [67]. Upregulation of bone morphogenetic protein-9 (BMP-9), an important member of the transforming growth factor (TGF)-β superfamily, dramatically induced MG-63 cell apoptosis due to reduced expression of PI3Kp85α and pAkt, suggesting that BMP-9 functions as a tumor suppressor in OS by inhibiting the PI3K/Akt signaling pathway [68]. A combination of radiation and arsenic trioxide (ATO) could deactivate the PI3K/Akt signaling, consequently enhancing induction of apoptosis in HOS cells [69]. Grifolin is a natural biologically active substance isolated from the edible bodies of the mushroom Albatrellus confluens. Treatment of U2OS and MG-63 with grifolin strong stimulated the release of cytochrome c accompanied by activation of caspase-9 and caspase-3 as well as downregulation of the inhibitor of apoptosis protein (IAP) due to a reduction in p-AKT levels, leading to increased cell death [70]. Taken together, these observations suggest that activation of the PI3K/Akt signaling pathway is able to inhibit apoptosis of OS cells, and targeting this pathway may represent a novel strategy to attenuate OS growth. Bcl-2 is the most important member of a family of proteins related to cell death signaling, and was first isolated as the product of an oncogene. The Bcl-2 protein family is composed of anti-apoptotic proteins such as Bcl-2 and Bcl-xL, and pro-apoptotic proteins such as Bax, Bak and Bad.

388 389

O

397 398

387

3.5. Angiogenesis

R O

3.4. Apoptosis

385 386

445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463

The mechanism involving the growth of novel blood vessels from the preexisting blood vessels is called angiogenesis. Angiogenesis not only plays an important role in physiological processes, but also contributes to the pathology of a number of cancers, including OS. It has been reported that nude mice injected with human OS 3AB-OS pluripotent CSCs, which were isolated from MG-63 cells, displayed incremental Akt levels and tumor blood vessel density [74]. Thus, activation of the PI3K/Akt pathway is important for angiogenesis of OS. Angiogenesis is a multifactorial and complex process. Among these factors, vascular endothelial growth factor (VEGF) is the most important [75]. VEGF can increase permeability of capillaries, promote migration of tip cells, and thus contribute to proliferation and survival of the stalk endothelial cells. A recent study by Wu et al. found that silencing of Ether à go-go 1 (Eag1), a central nervous system (CNS)-localized channel contributing to cancerogenesis, inhibited the activation of the VEGF/PI3K/Akt pathway and then decreased intratumoral microvessel density in a xenograft mouse model of OS [76]. Apart from VEGF, there are many other growth factors responsible for the angiogenic process. Hypoxia usually occurs as the tumor outgrows its blood supply, leading to excessive hypoxia-inducible factor-1 (HIF-1) production. HIF-1 can induce the secretion of multiple proangiogenic molecules, such as PDGF, TGF, basic fibroblast growth factor (bFGF). Several studies have shown that these growth factors exert a proangiogenic effect in OS progression [77–79]. As described above, they are also potent activators of the PI3K/Akt pathway. Therefore, activated PI3K/Akt pathway may play a critical role in OS angiogenesis induced by PDGF, TGF and bFGF. However, more mechanistic studies are necessary to elucidate the effects of this signaling cascade on the neovascularization of OS.

464

3.6. Metastasis

492

Tumor metastasis consists of a series of complex processes, including attachment of primary cancer cells to ECM components, invasion through the basement membrane, entry into the circulation, and extravasation to distal tissues [80]. OS is characterized by a high propensity for pulmonary metastasis, a major cause of death. Multi-agent chemotherapy increases the five-year overall survival rate of patients with localized disease to between 60% and 70%; however, those with pulmonary metastasis have a poor survival rate ranging from 11% to 20%. There is growing evidence that the PI3K/Akt pathway plays an important role in pulmonary metastasis of OS. It has been shown that Akt activity is significantly increased in OS tissues from 24 patients with pulmonary metastatic disease [81]. In a study regarding the association between AKT single nucleotide polymorphisms (SNPs) and OS

493

P

396

383 384

These proteins mainly modulate apoptosis at the mitochondrial outer membrane and control the initiation of mitochondrial outer membrane permeabilization [71]. Transfection of miR-223 mimic into MG-63 cells showed significant G0/G1 phase arrest and elevated apoptotic rate by directly targeting HSP90B1 [41]. Mechanistically, protein levels of PI3k, p-Akt, mTOR and Bcl-2 were decreased, whereas Bid levels were increased [41]. Phytoestrogens are known to block tumor progression by inhibiting proliferation and inducing apoptosis in cancer cells. Administration of 5,7-dihydroxy-4′-methoxyisoflavone, a phytoestrogen, markedly diminished p-Akt levels and subsequently stimulated downregulation of Bcl-2 expression and upregulation of Bax expression, which resulted in induction U2OS cell apoptosis [72]. Additionally, combined treatment with cisplatin plus celecoxib (an inhibitor of cyclooxygenase-2) suppressed activation of the PI3K/Akt signaling and in turn attenuated Bcl-2 expression, leading to a significant increase in the percentage of apoptotic MG-63 cells [73]. Thus, an increased ratio of Bcl-2 to Bid or Bax plays an important role in PI3K/Akt-induced prevention of OS cell apoptosis.

T

394 395

and CDK6 by disrupting active kinase complexes with cyclin D1 and have no impact on other CDKs. A study by Liu et al. found that exposure of HOS cells to human T-cell leukemia virus type 1 (HTLV-1) activated the PI3K/Akt pathway and then downregulated p21CIFI/WIFI and p27KIP1 expression, thus promoting cell cycle progression [60]. Additionally, activation of the PI3K/Akt pathway induced by cysteine-rich 61 (Cyr61), an ECM-associated growth factor, resulted in a subcellular redistribution of p21CIFI/WIFI, which could strong stimulated cell division and proliferation of Saos-2 cells [61]. In contrast, administration of matrine (a natural product) diminished Akt expression and subsequently enhanced p27KIP1 expression, which prevented cyclin D1 activity and arrested cell cycle at G0/G1 phase in MG-63, U2OS and Saos-2 cells [62]. However, whether the members of the INK4 family are involved in PI3K/Akt-mediated regulation of cell cycle in OS cells remains unknown, representing a promising field for future research.

D

381 382


E

6


465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491

494 495 496 497 498 499 500 501 502 503 504 505 506


528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564

568 569 570

C

526 527

E

524 525

R

4. Treatment targeting the PI3K/Akt pathway in OS

621

Although introduction of the neoadjuvant chemotherapy has shown efficacy in OS over the past decades, long-term survival rate remains low for OS patients, highlighting the need to search for new therapeutic approaches. As mentioned above, activation of the PI3K/Akt pathway plays a critically oncogenic role in the initiation and progression of OS. Thus, a number of compounds that can inhibit this pathway have been developed to treat OS or other malignancies (Table 1).

622

4.1. PI3K inhibitors

629

F

522 523

R

520 521

N C O

518 519

U

516 517

O

The adjuvant chemotherapy, such as cisplatin, doxorubicin, and methotrexate has greatly improved survival of OS patients. However, chemotherapy can produce chemoresistance and even lead to relapse or metastatic disease [97]. Currently, resistance to chemotherapy has

514 515

R O

567

513

571 572

P

3.7. Chemoresistance

511 512

become a major obstacle in the treatment of OS. Chemoresistance in OS appears to be mediated through multiple mechanisms, including less intracellular drug accumulation, drug inactivation, increased DNA repair, perturbations in signal transduction pathways, decreased apoptosis, autophagy, aberrant expression of miRNAs, and CSCs [98]. Interestingly, dysregulation of the PI3K/Akt pathway can influence chemosensitivity of OS cells. TWIST, also called TWIST1, belongs to the basic helix-loop-helix (bHLH) transcription factor family. Overexpression of TWIST has been shown to enhance sensitivity of MG-63 and Saos-2 cells to cisplatin by downregulating endothelin-1 (ET-1)/ endothelin A receptor (ETAR) signaling via inhibition of the PI3K/Akt pathway [99]. In contrast, knockdown of TWIST in MG-63 cells significantly elevated the soluble β-catenin levels and contributed to cell survival against cisplatin, which was reversed by β-catenin siRNA (small interfering RNA) or PI3K inhibitor LY294002 [100]. Genistein, a nontoxic flavonoid compound, has potent antitumor activities in multiple types of malignancies, including OS. OS cells treated with genistein exhibited a decreased resistance to gemcitabine through blocking Akt phosphorylation and NF-κB nuclear translocation [101,102]. Likewise, inhibition of calpain-6 was found to promote spontaneous apoptosis and increase sensitivity to doxorubicin and methotrexate in U2OS cells, which was attributed to partial inactivation of the PI3K/Akt/NF-κB pathway [103]. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1), a member of the polycomb group family of transcriptional regulators, was originally identified as an oncogenic partner of c-Myc in murine lymphomagenesis [104]. It has been proposed that downregulation of BMI-1 by lentivirus mediated RNA interference (RNAi) significantly impairs cell viability and colony formation in vitro and tumorigenesis in vivo of OS cells, and BMI-1 knockdown also sensitizes cells to cisplatin through inhibition of the PI3K/AKT pathway [105]. Autophagy is a homeostatic and evolutionarily conserved process that can degrade cellular organelles and proteins and maintain cellular biosynthesis, and it has been suggested as an important mechanism leading to chemoresistance of OS cells during clinical chemotherapy [98,106]. Chiu et al. reported that inhibition of the PI3K/Akt pathway by a combination of ATO and radiation induced autophagy and then enhanced sensitivity of HOS cells to ATO [69]. Dysregulation of miRNA has been shown to be implicated in tumorigenesis and development of multiple malignancies [107]. Several studies have demonstrated a link between altered PI3K/Akt signaling and drug-resistance in OS. Introduction of miR-221 mimic into human OS cell lines SOSP-9607 and MG-63 induced cell survival and cisplatin resistance by directly targeting PTEN, but these effects were abrogated in the presence of PTEN cDNA lacking 3′-UTR or PI3K inhibitor LY294002 [43]. In addition, a study by Chang et al. found that the miR-101 mimic interfered with autophagosome formation and then improved U2OS cell resistance to doxorubicin [108]. Given the negative regulation of miR-101 expression by PI3K/Akt signaling [109], it is likely that chemoresistance of OS cells mediated by the PI3K/Akt signaling is also associated with reduced miR-101. However, further research is required to test this possibility.

D

566

509 510

T

565

metastatic susceptibility in a Chinese population, patients carrying the genotype AA of AKT rs6973569 had a higher risk of OS metastasis [82]. TGF-β-inducible gene-h3 (βig-h3), also known as TGFBI, is widely expressed in various types of tumor cells. A recent study has demonstrated that βig-h3 can enhance the metastasis potential of Saos-2 cells via integrin α2β1-mediated PI3K/AKT signal pathway [83]. TGFα, one member of the EGF-like family, functions as an activator of the PI3K/Akt signaling. Knockdown of TGF-α inhibited the PI3K/Akt/NF-κB pathway and then downregulated intercellular adhesion molecule-1 (ICAM-1) expression, leading to a significant decrease in OS cell distant metastasis [84]. Nude mice treated with OS LM8 cells displayed strong pulmonary metastasis and higher p-Akt levels, and inhibition of the PI3K/Akt signaling using LY294002 (a PI3K inhibitor) or a dominant negative form of Akt markedly reduced pulmonary metastasis [85]. In addition to promotion of tumor cell invasion, MMPs are closely associated with distal metastasis of tumors. It has been demonstrated that inhibition of Akt by troglitazone decreased MMP-2 secretion and then retarded the development of pulmonary metastasis in nude mice implanted with LM8 cells on their backs [86]. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. In a xenograft mouse OS model, administration of YM529/ONO-5920 partially deactivated the Ras/PI3K/Akt signaling and in turn attenuated the expression and activities of MMP-1, MMP-2 and MMP-9, which led to reduced LM8 cell metastasis [87]. Collectively, increased MMP-1, MMP-2 and MMP-9 activities may be an important mechanism by which the PI3K/Akt pathway facilitates pulmonary metastatic nodule formation of OS. Ezrin, an important member of the ezrin-radixin-moesin (ERM) family of proteins, serves as a cross-linker between the actin cytoskeleton and the plasma membrane, thus allowing the cells to interact directly with its microenvironment. Ezrin not only possesses many normal physiological functions including maintenance of cell shape and polarity as well as regulation of cell growth and differentiation, but also has potent oncogenic effects [88–90]. Studies from our own and other groups have demonstrated that overexpression of ezrin promotes invasion, migration and metastasis of OS cells [91], but ezrin silencing or its small molecule inhibitors NSC305787 and NSC668394 protect against OS development [92,93]. A recent study by Krishnan et al. showed that inhibition of ezrin through the expression of a nonphosphorylatable T567A mutant slowed down primary growth of Ewing's sarcoma cells in vitro, and reduced the ability of tumor cells to form experimental metastases in vivo [94]. Mechanistically, the action of ezrin in Ewing's sarcoma was dependent on the AKT/mTOR signal transduction cascade [94]. These results imply that ezrin-mediated activation of the AKT/mTOR pathway may also contribute to the development of pulmonary metastasis in OS patients. However, further studies are needed to elucidate the effect. CXC-chemokine ligand 12 (CXCL12), as known as stromal cellderived factor-1 (SDF-1), can bind to its cognate receptors, CXCchemokine receptor (CXCR) 4 and 7, with high affinity. The interaction of CXCL12 with CXCR4 and CXCR7 stimulates the activation of several downstream signaling pathways, predominantly the PI3K/Akt pathway, thereby regulating tumor progression and metastasis [95]. Treatment with CXCL12 has been shown to promote the migration and adhesion of OS cells, whereas the development of pulmonary metastasis after injection of OS cells in a mouse model is significantly prevented by administration of T134 peptide, an inhibitor of CXCR4 [96]. Thus, it is possible that the PI3K/Akt pathway is involved in CXCL12-mediated pulmonary metastasis of OS.

E

507 508

7

573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620

623 624 625 626 627 628

There are two types of PI3K inhibitors, namely pan-PI3K and 630 isoform-specific inhibitors. The Pan-inhibitors, also known as the first- 631 generation of PI3K inhibitors, include compounds such as Wortmannin 632


Table 1 Experimentally verified compounds that can suppress the PI3K/Akt pathway in OS. Groups

Agents

Targets

Clinical trial stages

References

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27

PI3K inhibitors

Wortmannin LY294002 BYL719 BKM120 NVP-BEZ235

Class I PI3Ks Class I PI3Ks PI3Kα PI3Kα PI3K, mTOR

Phase 0 Phase 0 Phase 0 Phase 0 Phase 0

[110] [111,112] [113] [47] [115,116]

GSK690693 MK-2206 Perifosine Rapamycin Everolimus Temsirolimus Ridaforolimus PP242 5-azacytidine Tepoxalin Caffeine Oxymatrine Selaginella tamariscina Icariside II Quercetin Formononetin Curcumin

Akt Akt Akt mTORC1 mTORC1 mTORC1 mTORC1 mTORC1, mTORC2 PTEN PTEN PTEN PI3K, Akt PI3K, Akt

Phase 0 Phase 0 Phase 0 Phase 0 Phase 0 Phase I Phase II Phase 0 Phase 0 Phase 0 Phase 0 Phase 0 Phase 0

[120] [20] [31] [124,125] [126] [128] [129] [130] [132] [133] [134] [136] [137]

PI3K, Akt, mTOR Akt Akt Akt

Phase 0 Phase 0 Phase 0 Phase 0

[138] [139] [140] [141]

633

Natural compounds

657

4.2. Dual PI3K/mTOR inhibitors

658

Dual PI3K/mTOR inhibitors target the ATP site of all p110 isoforms of PI3K as well as both mTOR complexes, thereby more completely inhibiting the whole PI3K/AKT/mTOR signaling axis. NVP-BEZ235, a dual class I PI3K/mTOR inhibitor, is shown to suppress OS cell migration and metastasis, and combination with vincristine further potentiates its antimetastatic effects [115]. A later study by Gobin et al. has demonstrated that administration of NVP-BEZ235 can block OS cell growth both in vitro and in vivo [116]. GDC-0980 is another dual PI3K/mTOR inhibitor. In breast cancer, uterine serous carcinomas and non-small-cell lung cancer (NSCLC) cell lines with hyperactivated PI3K signaling caused by PIK3CA mutation or PTEN loss, GDC-0980 led to inhibition of cell growth and viability [117–119]. Moreover, GDC-0941 in

648 649 650 651 652 653 654 655

659 660 661 662 663 664 665 666 667 668 669

C

E

R

646 647

R

644 645

O

642 643

C

640 641

N

638 639

U

636 637

677 678

4.4. mTOR inhibitors

703

Given that mTOR is the most important downstream molecule of the PI3K/Akt pathway, mTOR inhibitors are also useful for blockade of the signaling cascade. Since its discovery in 1975, rapamycin has been extensively investigated as a strong mTOR inhibitor, because it can form a complex with FK506-binding protein (FKBP12), which then binds to C-terminal region of mTOR, interfering with the activation of mTORC1 but not mTORC2 [123]. Treatment of MG-63 cells with rapamycin resulted in a significant suppression of cell viability and enhanced cell death mediated by cis-diamminedichloroplatinum (CDDP) [124]. Additionally, in a murine OS cell line K7M2, rapamycin administration deactivated the mTOR/aldehyde dehydrogenase (ALDH, a stem cell marker) signaling and subsequently prevented cell proliferation, migration, and invasion [125]. However, similar to the first-generation of PI3K inhibitors, rapamycin has a number of pharmacological drawbacks as well. Thus, several more reliable rapamycin analogs including RAD001 (everolimus), CCI-779 (temsirolimus) and ridaforolimus have been developed. Everolimus and temsirolimus have shown cytostatic activity in preclinical models of OS and antitumor activity when used in combination with chemotherapeutic drugs [126,127]. Moreover, temsirolimus combined with irinotecan and temozolomide have entered phase I clinical trials in children with recurrent/refractory solid tumors including OS and show better antitumor efficacy, without obvious adverse safety concerns observed [128]. In a multicenter, open-label, single-arm, phase II trial evaluating the antitumor activity of ridaforolimus in 212 patients with distinct subtypes of advanced sarcomas, 61 patients (28.8%) achieved clinical benefit response, and median overall survival time was 40 weeks [129]. Evaluation Criteria in Solid Tumors (RECIST) has demonstrated that response rate is 1.9%, with four patients

704

T

656

(a fungal metabolite) and LY294002, which can bind all class I PI3Ks. Inhibition of autophagy by Wortmannin has been reported to enhance MG-63 cell apoptosis induced by dendropanoxide, which is newly isolated from leaves and stem of Dendropanax morbifera Leveille [110]. Extensive data have found that administration of LY294002 can reverse aggressive biological behavior of OS cells [111,112]. Despite effective inhibition of the PI3K pathway and demonstration of anti-OS activity, these pan-inhibitors may be never fully developed as anticancer drugs for clinical use because of very poor pharmacokinetic properties, including weak solubility, instability, and high toxicity. To improve pharmacokinetic properties, isoform-specific inhibitors that are able to selectively inhibit PI3K p110α, β, δ or γ catalytic subunits have emerged. This greater specificity offers the possibility of reduced side effects, thus allowing a higher tolerated dose. BYL719 is a new specific PI3Kα inhibitor that blocks the ATP site. It is suggested that BYL719 attenuates cell proliferation by blocking cell cycle in G0/ G1 phase without obvious effects on apoptosis in HOS and MOS-J cells, and blocks tumor ectopic bone formation in murine preclinical models of OS [113]. BKM120 is also a PI3Kα inhibitor. MG-63 cells treated with BKM120 displayed a significant decrease in cell invasion and viability [47]. Inhibitors of PI3Kβ, such as GSK2636771, are in clinical development, and a phase I/IIa, first-in-human study is currently conducted in patients with PTEN-deficient advanced solid tumors [114]. However, it remains unclear whether GSK2636771 has anti-OS effects.

634 635

As a critical signaling junction downstream of PI3K, AKT provides another clear target for blockade of PI3K signaling. In recent years, a number of allosteric and ATP-competitive Akt inhibitors have been developed. ATPcompetitive AKT inhibitors belong to small molecule inhibitors that can inhibit three isoforms of Akt, acting through ATP competition, with specificity limited to Akt. It has recently been demonstrated that the ATPcompetitive AKT inhibitor GSK690693 decreases OS cell proliferation in vitro, and inhibits tumor growth and increases survival rate in a xenograft mouse OS model [120]. Allosteric Akt inhibitors can bind to the PH domain of Akt, thereby promoting an inactive form of the enzyme by preventing localization to the membrane or access to the PDK-1 dependent phosphorylation site. MK-2206 is a small molecule allosteric Akt inhibitor that has entered phase II clinical trials and shown marked suppression of breast cancer growth with acceptable tolerability [121]. A recent study observed that treatment with MK-2206 also reduced the proliferation of U2OS and HOS cells [20]. Likewise, administration of perifosine, another allosteric Akt inhibitor, induced cell apoptosis and growth inhibition in cultured human OS cells by blocking the Akt/mTORC1 signaling and stimulating the activation of caspase-3, c-Jun N-terminal kinases (JNK) and p53 [31]. Furthermore, a low dose of perifosine was found to enhance sensitivity of OS cells to etoposide or doxorubicin [31]. The most common adverse reactions of this oral drug are fatigue and gastrointestinal toxicity including nausea, vomiting, diarrhea and abdominal pain. At present, perifosine is in phase II clinical trials for patients with Waldenström's macroglobulinemia, a rare and low-grade lymphoproliferative disorder [122].

O

PTEN activators

676

R O

mTOR inhibitors

4.3. Akt inhibitors

P

Dual PI3K/mTOR inhibitors Akt inhibitors

670 671

F

t1:3

combination with chemotherapeutic drugs such as paclitaxel or erlotinib had greater effects on NSCLC cell viability than GDC-0941 alone [119]. Given the oncogenic role of the PI3K/mTOR pathway in OS, addition of GDC-0941 might be beneficial to control OS progression. However, further studies are necessary to determine whether these two inhibitors are efficacious in patients with OS.

D

t1:1 t1:2


E

8


672 673 674 675

679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702

705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731


759

4.6. Natural compounds from plants

760

In the development of bioactive chemical, natural products have a rich and long history. The traditional Chinese medicinal herbs, as an important novel source with a wide range of pharmaceutical potential, are being used to treat human ailments including almost all types of tumors [135]. Some of which have shown promise for OS therapy through inhibition of the PI3K/Akt pathway. For example, oxymatrine, classified as a quinolizidine alkaloid, is a phytochemical product derived from Sophora flavescens. More recently, Zhang et al. reported that addition of oxymatrine to human OS MNNG/HOS cells strongly induced mitochondria-dependent apoptosis via stimulating the dephosphorylation of PI3K and Akt [136]. These authors also found that oxymatrine significantly inhibited tumor growth in female Balb/C nude mice bearing MNNG/HOS xenograft tumors, without obvious side effects [136]. Selaginella tamariscina is also a traditional medicinal plant that has a potential therapeutic benefit for some advanced cancer patients in the Orient. Treatment with Selaginella tamariscina (0–50 μg/mL) concentration-dependently prevented the migratory and invasive capacities of U2OS cells without cytotoxic effects, which was attributed to decreased MMP-2 and MMP-9 secretion by blocking the phosphorylation of PI3K and Akt [137]. Icariside II, one of the most important natural flavonoids, is extracted from Horny Goat Weed (Epimedium koreanum Nakai). Administration of Icariside II has been shown to deactivate the PI3K/Akt/mTOR pathway and then diminish OS cell proliferation as well as transplantable tumor growth in sarcoma-180 bearing mice [138]. Application of quercetin, another polyphenolic flavonoid isolated from plants, significantly reduced cell viability and promoted apoptosis, which was accompanied by a marked decrease of Akt activity [139]. Formononetin, a major isoflavones in Radix astragali, could trigger apoptosis of U2OS cells in vitro, through Akt inactivation along with downregulation of Bcl-2 and upregulation of Bax [140]. Additionally, curcumin-loaded nanoparticles markedly decreased p-Akt levels, which resulted in induction of anti-proliferative effects and apoptosis in U2OS cells [141]. Together, these observations suggest

761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792

796

Disclosure

T

C

756 757

E

754 755

R

752 753

R

750 751

N C O

748 749

U

746 747

It is clear that the PI3K/Akt signaling pathway is a critical driver of oncogenesis in OS. This complex pathway has been taken into consideration as one of the most attractive targets for the development of antiOS agents. However, in addition to mTOR inhibitors, other inhibitors have not entered clinical trials in OS. Further investigation will be necessary and urgent to evaluate their applicability in the clinical settings. At presently, some challenges remain existed in the treatment accurately targeting the PI3K/Akt signaling pathway. The biggest obstacle is devoid of reliable predictive biomarkers that can identify patients who will be most likely to benefit from these types of therapy. Another issue is the failure in generating a lasting outcome, because cancer cells are able to compensate for the inhibitory effects of this pathway and thereby acquire treatment resistance through different feedback loop and cross-talk mechanisms. The future development of these promising inhibitors should thus focus on combined strategies, including the concomitant or sequential blockade of signaling pathways. Lastly, it is essential to minimize the side effects of these drugs for clinical application. Understanding these questions will increase our chances to design novel therapeutic interventions targeting the PI3K/Akt pathway to help further reduce the mortality of OS patients in the future.

F

758

PTEN acts as a main negative regulator of the PI3K/Akt pathway, and the loss of PTEN activity is frequently discovered in OS [131]. Thus, PTEN activators may be an alternative approach for suppression of the pathway in OS. Indeed, treatment with 5-azacytidine significantly upregulated PTEN expression and then induced apoptosis in MG-63 cells [132]. In agreement, downstream Akt signaling was deactivated by 5azacytidine [132]. Tepoxalin is an inhibitor of 5-lipoxygenase (5-LOX), and has been shown to slow down canine OS xenograft growth. Administration of tepoxalin could increase PTEN activity by preventing its alkylation or oxidation, leading to Akt inhibition and induction of apoptosis in canine OS cell lines [133]. Also, addition of caffeine to HOS cells promoted PTEN-mediated Akt blockade and in turn attenuated tumor cell proliferation [134]. Together, although not historically thought of as a druggable target in the PI3K/Akt pathway, current research focusing on PTEN upregulation for OS treatment is ongoing.

739 740

795

O

744 745

738

5. Conclusions and future directions

R O

4.5. PTEN activators

736 737

P

743

734 735

that these herbs may be powerful candidates to develop novel preven- 793 tive agents for OS cases. 794

The authors have declared no conflict of interest.

E

741 742

achieving confirmed partial response, including two cases with OS [129]. Related adverse events are generally mild or moderate and consist primarily of stomatitis, mucosal inflammation, mouth ulceration, rash and fatigue [129]. A phase III trial based on these data will further define the antitumor activity of ridaforolimus in OS. Despite the promise of these mTORC1 allosteric inhibitors, feedback activation of Akt persists via mTORC2, potentially shifting the focus to ATP-competitive compounds that can inactivate both complexes and completely abrogate mTOR signaling. The most important inhibitor of this class is PP242, and it can effectively attenuate OS cell (MG-63, U2OS and Saos-2) migration and promote cisplatin-induced apoptosis [130].

D

732 733

9

797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817

Acknowledgment

818

The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (31400802), and the Natural Science Foundation of Hunan Province, China (2015JJ4043 and 2015JJ5003).

819 820

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1113 1114 1115 Q6 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 Q7 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196

Akt signaling pathway.

4-Hydroxy-2-nonenal induces apoptosis by inhibiting AKT signaling in human osteosarcoma cells.

MMP9 signaling pathway.

β-catenin signaling.

AKT signaling pathway.

Akt signaling pathway.

TRAF4 enhances osteosarcoma cell proliferation and invasion by Akt signaling pathway.

mTOR signaling.

Deciphering signaling networks in osteosarcoma pathobiology.

GSK3β signaling pathway.

NF-κB signaling pathway.

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

Curcumin-loaded nanoparticles enhance apoptotic cell death of U2OS human osteosarcoma cells through the Akt-Bad signaling pathway.

AKT signaling pathway.

Kinome and mRNA expression profiling of high-grade osteosarcoma cell lines implies Akt signaling as possible target for therapy.

AKT but not p38MAPK signaling pathway.

mTOR Signaling in Cancer.

AKT signaling in ERBB2-amplified breast cancer.

Aberrant hedgehog signaling and clinical outcome in osteosarcoma.

Involvement and targeted intervention of dysregulated Hedgehog signaling in osteosarcoma.

Hedgehog signaling inhibitors as anti-cancer agents in osteosarcoma.

YAP signaling pathway is involved in osteosarcoma chemoresistance.

When mTORC2-AKT signaling meets cell polarity.

Akt signaling.