Natural compounds targeting major cell signaling pathways: a novel paradigm for osteosarcoma therapy
Angulo et al. Journal of Hematology & Oncology
Natural compounds targeting major cell signaling pathways: a novel paradigm for osteosarcoma therapy
Pablo Angulo 1 2
Gaurav Kaushik 1
Dharmalingam Subramaniam 0 1
Prasad Dandawate 1
Kathleen Neville 3
Katherine Chastain 1 2
Shrikant Anant 0 1
0 The University of Kansas Cancer Center, The University of Kansas Medical Center , Kansas City, KS 66160 , USA
1 Department of Surgery, The University of Kansas Medical Center , 3901 Rainbow Boulevard, Mail Stop 3040, Kansas City, KS 66160 , USA
2 Division of Hematology and Oncology, Children's Mercy Hospital , Kansas City, MO 64108 , USA
3 Division of Hematology and Oncology, Arkansas Children's Hospital , Little Rock, AR 72202 , USA
Osteosarcoma is the most common primary bone cancer affecting children and adolescents worldwide. Despite an incidence of three cases per million annually, it accounts for an inordinate amount of morbidity and mortality. While the use of chemotherapy (cisplatin, doxorubicin, and methotrexate) in the last century initially resulted in marginal improvement in survival over surgery alone, survival has not improved further in the past four decades. Patients with metastatic osteosarcoma have an especially poor prognosis, with only 30% overall survival. Hence, there is a substantial need for new therapies. The inability to control the metastatic progression of this localized cancer stems from a lack of complete knowledge of the biology of osteosarcoma. Consequently, there has been an aggressive undertaking of scientific investigation of various signaling pathways that could be instrumental in understanding the pathogenesis of osteosarcoma. Here, we review these cancer signaling pathways, including Notch, Wnt, Hedgehog, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT, and JAK/STAT, and their specific role in osteosarcoma. In addition, we highlight numerous natural compounds that have been documented to target these pathways effectively, including curcumin, diallyl trisulfide, resveratrol, apigenin, cyclopamine, and sulforaphane. We elucidate through references that these natural compounds can induce cancer signaling pathway manipulation and possibly facilitate new treatment modalities for osteosarcoma.
Osteosarcoma; Signaling pathways; Natural compounds; Ezrin
Osteosarcoma (OS) is the most commonly diagnosed
primary bone malignancy, with an incidence of 0.2–3 cases/
100,000 annually in children and 0.8–11 cases/100,000 in
adolescents. The incidence peaks in the second decade of
life. While only 20% patients present with metastasis that is
clinically detectable, the majority of the remaining 80% are
presumed to have undetectable micro-metastases at
diagnosis . The cancer can be found on the bone surface,
within the bone, or in extraosseous sites, including the
lung . The etiology of OS remains uncertain despite
advancements in molecular sciences. The only known
environmental factor for OS is ionizing radiation .
OS is ranked among the leading causes of cancer
mortality in the pediatric population . Therapy includes
preoperative chemotherapy, surgery, and postoperative
chemotherapy (cisplatin, methotrexate, and
doxorubicin). Additional chemotherapy (ifosfamide and
etoposide) has been reserved for patients with high risk of
metastatic disease. While chemotherapy has increased
overall survival to 60–75%, survival rates have
remained the same for the last 30–40 years .
Moreover, only 30% patients with metastatic OS achieve a
5-year event free survival .
The vast majority of OS appears to be sporadic,
occurring in patients without common familial members
affected. Nevertheless, there is growing support that the
cancer is associated with the activation of numerous
oncogenes, including cyclin D1, mouse double minute 2
homolog (MDM2), and c-Myc. Additional studies have
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demonstrated that various signaling pathways appear to
be involved in the tumorigenesis of OS, including Wnt
and PI3/Akt . Hence, characterizing molecular targets
that are specific for OS will be paramount for developing
new strategies for treatment modalities.
Our group has been investigating key molecular
signaling pathways that are integral in the origin,
proliferation, and survival of osteosarcoma cells. Genes in these
pathways are often mutated, resulting in activated cancer
stem cells that proliferate without the normal regulatory
mechanisms seen in noncancerous cells. In this review,
we summarize a variety of signaling pathways that have
demonstrated important roles in OS pathogenesis. In
addition, we review numerous phytochemicals and
inhibitors targeting these signaling pathways that show
promising treatment abilities in OS. Table 1 lists these
important cell signaling pathways along with the
respective specific inhibitors. Figure 1 outlines the
chemical structures of the six compounds described
including curcumin, diallyl trisulfide, resveratrol, apigenin,
cyclopamine, and sulforaphane. Through continuous
research of these various pathways, an improved
understanding of the molecular machinery promoting OS can
be attained. Successful future treatment modalities depend
on our ability to better understand and target these
The Notch signaling pathway is an evolutionarily
conserved pathway that plays an important role in embryonic
and postnatal development in many organisms. The
pathway is highly pleiotropic and affects vital processes of
organ development as well as regulation of self-renewal of
adult stem cells, thereby maintaining tissue homeostasis
. However, due to its multifunctional nature, this
pathway is vulnerable to aberrant activation of signaling
components and is associated with multiple human disorders,
including various developmental syndromes and
malignancies [8–10]. Therefore, in recent years, notch signaling
has become one of the most important potential targets
for developing novel therapeutic strategies.
Notch signaling is a multi-tiered, well-organized, tightly
regulated cell/tissue-dependent cascade of signaling events.
It requires various components for its maturation,
activation, and execution. The Notch signaling family consists of
four receptors, known as Notch-1 to Notch-4, and five DSL
(Delta/Serrate/Lag-2) ligands, known as Jagged-1 and
Jagged-2 (Jag-1 and Jag-2) and Delta-like-1, Delta-like-3,
and Delta-like-4 (Dll-1, Dll-3, and Dll-4).
Both the receptors and the ligands transmembrane
proteins, and activation of the pathway occurs when a
ligand from the neighboring cell interacts with the receptor
[8, 11]. The interaction triggers conformational changes in
the ligand-receptor complex that exposes an extracellular
site on the notch receptor to proteolytic cleavage by tumor
necrosis factor-alpha converting enzyme (TACE/ADAM17/
CD156q), a component of the “a disintegrin and
metalloprotease”, or ADAM (Fig. 2). A key regulatory step in notch
activation and signaling, this cleavage generates the
membrane-attached notch extracellular truncation (NEXT)
fragment, which is located within the negative regulatory
region (NRR) of the extracellular domain of the notch
receptor. This NEXT fragment acts as a substrate for the
γsecretase protein complex, which consists of nicastrin,
presenilin, presenilin enhancer-2 (PEN-2), and anterior
pharynx defective 1 (APH1) [12, 13]. Once it is generated,
NEXT is cleaved by γ-secretase to release and trigger
Table 1 Effect of Natural Compounds Targeting Major Stem Cell Signaling Pathways
Downregulates transcription and
translation Notch-1 and downstream
genes Hes-1, Hey-1, and Hey-2
Diallyl trisulfide Targets Notch-1 intracellular domain
1Induces apoptosis by increasing reactive oxygen species
Decreases expression of Notch downstream genes. Increases
expression of potential tumor suppressor micro RNAs
(miR143 and miR-145) and decreases tumor promoting micro
Apoptosis of OS cells by decreasing mRNA and protein
expression of β-catenin and c-Myc
Histone H2AX phosphorylation causes telomere instability
and DNA damage
Decreases protein expression of β-catenin and decreases
matrix metalloproteinase 14 (MMP14) expression
Prevents signal transduction to GLIS
Suppresses ERK and AKT phosphorylation, induces apoptosis 
through G2/M phase arrest
Fig. 1 Chemical structure of the phytochemicals
Fig. 2 Notch signaling pathway. Ligand from the presenting cell
binds to the notch receptor on the receiving cell. Notch extracellular
truncated (NEXT) domain is cleaved by ADAM metalloprotease and
γ-secretase yielding the notch intracellular domain (NICD). NICD is
translocated to the nucleus where it complexes with transcription
factor CSL 9 CBF1/suppressor of hairless/Lag 1 and transcriptional
coactivator of the mastermind-like proteins (MAML). The complex
can then activate target gene transcription. Diallyl trisulfide (DATS)
treatment increases expression of tumor suppressor microRNAs:
miR-143 and miR-145. MicroRNAs bind to Notch1 mRNA and results in
mRNA degradation with no translation of Notch1 protein. Curcumin
downregulates transcription and translation Notch1 and downstream
genes Hes-1, Hey-1, and Hey-2 of the nucleus
translocation of the notch intracellular domain (NICD) into
the nucleus. The active NICD can bind to mastermind-like
proteins (MAML) and recombination signaling binding
protein of hairless-J (RBPJ/CBF1) and form a nuclear
activator complex to regulate transcription of downstream
gene targets such as the hairy and enhancer of split (Hes)
family of genes and the Hes-related family BHLH
transcription factor with YRPW motif (Hey) family genes (Fig. 2). In
the absence of NICD, RBPJ/CSL may associate with
corepressor proteins and repress transcription of target genes
Studies have documented the association of the notch
signaling pathway with the resistance, aggressiveness,
and metastatic potential of OS. This association has
been validated in various experimental models including
OS human/mouse cell lines, in vivo mice/canine models,
and also in patient samples. All of these studies showed
that OS cells with higher metastatic potential have
higher basal levels of notch receptors, especially notch-1,
notch-2, notch ligands (Dll-1/Jagged-1), and notch target
genes such as hey-1 and hes-1, as compared to normal
osteoblasts or non-metastatic OS cell lines. Higher
expression levels of these notch signaling associated genes
or proteins were shown to be involved in invasiveness
and metastasis and thus in impacting OS patient survival
[16, 17]. Increased levels of jagged-1 in OS cells promote
bone metastasis by activating stromal notch signaling.
IL-6 secretion from osteoblasts continues to augment
tumor growth . Notch signaling was also reported to
be associated with increased aldehyde dehydrogenase
(ALDH) activity, which results in an aggressive
metastatic phenotype in murine OS cell lines (K7M2 and
K12). K7M2 cells (highly metastatic in nature) showed
upregulation of expression of notch signaling genes,
including notch-1, notch-2, notch-4, and downstream
targets genes, such as stat-3 and hes-1, compared to K12
cells. Elevated ALDH activity in K7M2 cells was
abolished by inhibiting the notch signaling pathway and
hence resulted in decreased metastatic behavior .
Therefore, abolishing expression of hey-1, hes-1, notch-1,
and jagged-1 by using γ-secretase inhibitors (GSI) also
abolished their direct/indirect effects on survival, bone
metastasis, and invasiveness in OS [16, 18]. These findings
suggest that inhibiting notch signaling at various points
may be a novel therapeutic strategy for preventing OS
invasiveness and metastasis .
In the last decade, several studies have demonstrated the
role of microRNAs (miRNAs) in the progression,
differentiation, and function of different cell types and in the
pathogenesis of various human diseases. Recently, the expression
pattern of miRNAs and their role in osteosarcoma was
studied. It was observed that there was a significant
increase in expression of some miRNAs (10-fold) in OS
patients as compared to normal controls. Three of these
miRNAs (miR-338-3p, miR-891a, and miR-199b-5p) were
upregulated in OS cells. Further, ectopic expression of
inhibitor of miR-199b-5p in OS cell lines showed a change
in expression of notch pathway components and revealed
that miR-199b-5p plays a role in notch signaling in OS .
Further work demonstrated that the expression of the
microRNA 34 cluster (noted to downregulate Dll-1, notch
1, and notch 2) showed an inverse correlation with
invasiveness in some osteosarcoma tumors, suggesting that this
family of microRNAs may also be responsible for regulating
notch expression in some tumors . Additional studies
have shown an association with Wnt signaling in the
regulation of notch signaling in OS. In one study, Wnt10b
expressing U2OS human OS (U2OS-Wnt10b) cells were
compared to parental U2OS cells. In addition, differential
expression of 1003 genes was compared. Genes involved in
notch signaling (especially notch-1 and Jagged-1) were
upregulated, whereas the notch inhibitor was significantly
downregulated, leading to activation of the classic notch
responsive genes (hes-1 and hey-1) . These findings
suggested that activation of Notch signaling plays a critical
role in the pathogenesis of human OS and its inhibition
could be a therapeutic approach for the treatment of this
mesenchymal tumor .
The Wnt signaling pathway is a highly conserved pathway
responsible for a variety of functions including cell
migration, cell fate determination, organogenesis, and stem cell
renewal. Wnt signaling activates numerous transduction
cascades in the cell. These cascades include Wnt/β-catenin
dependent pathway and β-catenin-independent pathways.
Alterations and dysregulation in the Wnt pathway can
result in cancers of the skin, breast, and colon.
β-catenin is an integral protein in the Wnt signaling
pathway that is responsible for regulating gene
transcription and cell-to-cell adhesion. The level of the protein is
stably maintained through degradation and
phosphorylation. Mutations in β-catenin cause amino acid
substitutions resulting in inappropriate phosphorylation of the
protein. The phosphorylated protein is subsequently not
recognized appropriately by the ubiquitin ligase E3.
Hence, dysregulation of the Wnt pathway results in
βcatenin accumulating without being degraded and then
translocating to the nucleus where it activates
transcription of oncogenes . Expression of downstream genes
includes c-Myc, cyclin D1, and survivin (an inhibitor of
apoptosis). Wnt glycoproteins bind to the extracellular
transmembrane Frizzled receptor family. Thereafter, the
signal activates the protein Dishevelled (Dsh/DV1) in the
cytoplasm. Wnt can then branch into three different
signal cascades: canonical, non-canonical planar cell polarity,
and non-canonical Wnt/Ca2+ . The hallmark of the
canonical pathway is the translocation of β-catenin from
the cytoplasm to the nucleus where it acts as a coactivator
of transcription factors of TCF/LEF family (Fig. 3b).
Without the Wnt glycoprotein binding to the Frizzled receptor,
β-catenin would be degraded by a β-catenin destruction
complex. This degradation complex results in the
phosphorylation of β-catenin at various sites mediated by the
scaffolding protein Axin which can interact with glycogen
synthase 3β (GSK3), Casein kinase 1 alpha 1 (CK1α), and
β-catenin. The phosphorylation of β-catenin comes by
way of CK1α at serine 45 and by GSK3 at threonine 41,
serine 37, and serine 33. Those final phosphorylation sites
at serine 33 and 37 form a binding site for beta-transducin
repeat-containing E3 ubiquitin protein ligase (β-Trcp)
which can then degrade β-catenin  (Fig. 3a). The
hallmark of planar cell polarity is actin cytoskeleton
regulation. This pathway is responsible for organizing sensory
cilia of the inner ear as well as organizing hair follicles.
The crux of the Wnt/Ca2+ pathway is the stimulation
of intracellular calcium release from the endoplasmic
reticulum by way of interaction with G proteins. This
pathway is important for dorsal axis formation and
regulation of tissue separation. β-catenin is not
involved in either non-canonical pathway .
Wnt is known to play an important role in
osteoblastogenesis. Because osteosarcoma cancer cells are believed to
be derived from osteoblasts, it is reasonable to postulate
that antagonizing the Wnt pathway might yield inhibition
of osteosarcoma cells as osteoblastogenesis is impaired.
Wang et al. reported that the chemotherapeutic docetaxel
could successfully inhibit the proliferation of two
osteosarcoma cancer cell lines, U2OS and SaOS-2, in a
timedependent and dose-dependent manner by interfering
with the Wnt pathway. Docetaxel functioned by inhibiting
the transcriptional activity of β-catenin . Zhao et al.
also demonstrated that the Wnt pathway could be
targeted by utilizing naked cuticle homolog-2 gene
(NKD2), which encodes a protein that serves as a negative
Fig. 3 Wnt signaling pathway. a In the absence of the Wnt glycoprotein, β-catenin is degraded after being ubiquitinated and phosphorylated by
the destruction complex. Target genes in the nucleus are not activated. b In the presence of Wnt, the glycoprotein binds to the extracellular
transmembrane Frizzled receptor family (Fz and LRP5/6). Thereafter, the signal activates the protein Dishevelled (Dsh/DV1) in the cytoplasm. This
binding results in disrupting the β-catenin destruction complex of various proteins including: axin, casein kinase 1α, adenomatous polyposis coli
(APC), protein phosphatase 2A (PP2A), and glycogen synthase kinase 3 (GSK–3β). β-catenin translocates to nucleus where it can act as transcriptional
coactivator of transcription factors of TCF/LEF family. Resveratrol and apigenin decrease protein expression of β-catenin
regulator in the Wnt pathway. In a mouse model, NKD2
was overexpressed with osteosarcomas, and the ability of
the cancer cells to proliferate, invade, and metastasize was
markedly decreased. Evaluation of the tumors with NKD2
overexpression revealed downregulation in molecules
required for angiogenesis and upregulation of tumor
suppressor genes .
Wnt signaling pathway has also demonstrated to be
involved with the oncogene v-maf avian
musculoaponeurotic fibrosarcoma oncogene homolog K (MAFK), a
homolog that is integral in cell proliferation in vitro.
Using a gene microarray, Wang et al. showed that the
oncogene expression level of MAFK could be induced
by Wnt-1. Hence, the Wnt-1 induction of the expression
of MAFK resulted in a significant increase in the cell
viability, further demonstrating the role of Wnt in
osteosarcoma pathogenesis . These experiments provide
evidence that antagonizing the Wnt pathway could have
some therapeutic efficacy in osteosarcoma treatment.
It has been estimated that 70% of OS specimens possess
hedgehog (Hh) signaling components. Hedgehog was
discovered as a critical factor in the development and
progression of multiple cancers. The pathway is unique in that it is
comprised of both tumor suppressor genes and oncogenes.
The signaling pathway is associated with three ligands:
Sonic hedgehog (SHh), Indian hedgehog (IHh), Desert
hedgehog (DHh), and additional components of the
pathway include 12-transmembrane Patched proteins
(PTCH1 and PTCH2), 5-zinc finger transcription factors
GLI1, GLI2, GLI3 (glioma-associated oncogene homologs),
and the 7-transmembrane protein smoothened (SMO). In
the canonical pathway (beta catenin dependent), a ligand
will bind to PTCH1 (a transmembrane receptor), which
relieves SMO (G-protein coupled receptor-like protein).
SMO in turn can activate downstream transcription factors
called GLI family zinc finger proteins (Fig. 4b). If ligands
are not present, PTCH will block the entry of SMO. As a
result, SMO is not able to functionally inhibit protein
kinases including PKA, GSK-3b, and CK1. Hence, the
protein kinases can phosphorylate GLI proteins in complex
with SUFU and cause proteolytic cleavage of GLI. The
cleavage of GLI results in the formation of a repressed form
of GLI that will translocate to the nucleus and turn off
signaling (Fig. 4a). The usual Hh target genes include
transcription factors such as cyclin D1, B cell CLL/lymphoma 2
(BCL2), and vascular endothelial growth factor (VEGF). Lo
et al. evaluated Hh pathway in 42 human OS samples and
found higher expression levels of genes encoding IHH,
PTCH1, and GLI genes in the tumors. It is speculated that
high levels of IHh result in a larger tumor size .
Other research has demonstrated that SMO and GLI
activation are vital for OS progression. Hirotsu et al.
determined that SHh, DHh, PTCH1, GLI1, GLI2, and SMO
were overexpressed in five different OS cell lines (NHOst,
143B, HOS, MG63, and NOS-1). It was speculated that
the promoter of GLI1 was inactivated in human OS
Fig. 4 Hedgehog signaling pathway. a In the absence of Hh ligand, PTCH prevents activation of SMO. SMO cannot inhibit protein kinases
including PKA, GSK-3β, and CK1. These protein kinases phosphorylate GLI protein in complex with SUFU resulting in cleavage of GLI into a repressed
form. The repressed form will translocate to the nucleus inhibiting Hh target gene expression. b Hh ligand binds PTCH1 (transmembrane receptor).
Smoothened (SMO) is relieved and inhibits proteolytic cleavage of GLI protein resulting in an active form. The active GLI protein translocates to nucleus
and activates transcription factors. Cyclopamine binds to SMO preventing signal transduction to GLIS
specimens and that GLI2 mediated the activity of
downstream SMO; thus, GLI1 is downregulated in OS while
GLI2 is upregulated . Nagano et al. showed that GLI2
was involved in the tumor invasion and metastasis. In
mice that had GLI2 knocked down via transfection of
GLI2-shRNA, tumor growth was decreased compared to
mice that did not have GLI2 knocked down .
Drug discovery for the Hh pathway has been
concentrated on targeting SMO, which serves as the primary
transducer in Hh signaling. When SMO is inhibited, the
transcription factors GLI1 and GLI2 remain inactive. This
inactivity prevents the expression of tumor-activating
genes. As a result of inhibiting SMO, apoptosis and
growth arrest of OS cells occur in vivo and in vitro .
Hh inhibitors including the plant-derived cyclopamine
and its respective derivatives such as saridegib and
vismodegib are potentially promising drug options. However,
more research is needed to explore the broad biological
effects of inhibiting the Hh pathway .
PI3K-AKT-mTOR and Ras-Raf-MEK-ERK pathways
PI3Ks are a lipid kinase family that can be categorized into
three different classes based on homology and the
particular substrate they bind. Class I lipid kinase is most often
associated with cancer and is a heterodimer consisting of a
regulatory subunit and a catalytic subunit. When the
catalytic subunit is activated, phosphatidylinositol
4,5-biphosphate (PIP2) is altered to phosphatidylinositol
3,4,5triphosphate (PIP3). PIP3 is then able to recruit signaling
proteins including phosphoinositide-dependent kinase 1
(PDK1) and AKT. AKT is partially activated by PDK1 at
threonine 308 (Thr308). Thereafter, AKT is fully activated
at serine 473 (Ser473) by the following proteins:
integrinlinked kinase (ILK), DNA-dependent protein kinase
(DNAPK), mTORC2, and even AKT itself. Now fully activated,
AKT can translocate to the nucleus from the membrane
and the cytoplasm, where it can phosphorylate or activate
downstream targets  (Fig. 5).
Fig. 5 PI3K-AKT-mTOR and RAS-RAF-MEK-ERK pathways. Growth factor
binds to epidermal growth factor receptor and can proceed by two
different pathways. For the PI3 pathway, the ligand activates tyrosine
kinase receptor activity resulting in phosphorylation of receptor. PI3K
binds to phosphorylated receptor and becomes activated. PI3K then
binds to PIP2 on inner membrane and phosphorylates PIP2 to PIP3.
PIP3 activates AKT via PDK1. AKT can then phosphorylate and activate
protein mTOR which results in cell growth, cell proliferation, and cell
survival. For the RAS-RAF pathway, growth factor binding to tyrosine
kinase receptor activates RAS which in turn activates RAF. RAF activates
MEK which phosphorylates ERK to decrease apoptosis and increase cell
proliferation and growth. The compound sulforaphane suppresses the
phosphorylation of AKT and ERK
A key downstream target of the PI3/AKT pathway is
mammalian target of rapamycin (mTOR). The mTOR is a
serine/threonine kinase that regulates protein synthesis and
cell cycle progression. mTOR has two forms known
respectively as mTORC1 and mTORC2. mTORC1 controls
autonomous growth, while mTORC2 mediates cell survival
and proliferation. mTORC1 is integral in the carcinogenesis
of a multitude of cancers including OS. mTORC1 is made
of proline-rich AKT substrate (PRAS40), DEP
domaincontaining mTOR-interacting protein (Deptor), a
regulatory-associated protein of mTOR (Raptor), and
mammalian LST8/G-protein β-subunit like protein
(GβL). When activated, mTORC1 can mediate the
phosphorylation of ribosomal protein S6 kinases (S6K)
as well as the eukaryotic translation initiation factor
4E-binding protein 1 (4E-BP1). Phosphorylation of
4EBP1 causes the release of eukaryotic translation initiation
factor 4E (eIF4E), which leads to the translation of protein
as well as cell cycle progression . mTORC2 consists of
mTOR, LST8/G-protein β-subunit-like protein (GβL),
Rictor, and mammalian stress-activated protein
kinaseinteracting protein (mSIN1) and is an activator of AKT
enabling cell survival . mTORC2 was found to also
directly phosphorylate PI3K. This phosphorylation is
necessary to maximize the activation of the anti-apoptosis
kinase and enhance cell proliferation, migration, and
survival. This discovery has led to the development of small
molecular mTOR inhibitors . The tumor suppressor
tuberous sclerosis complex (Tsc) 2 can be phosphorylated
by AKT. This phosphorylation inhibits the formation of
Tsc1/Tsc1 heterodimers. Inhibition of Tsc1/Tsc2
heterodimers preserves an active GTP-bound state of the protein
Rheb (Ras homolog enriched in brain) and leads to an
increase in mTORC1 activity .
Other additional important targets that are affected by
AKT include the following: GSK3β, nuclear factor-κB
(NFκB), forkhead box O1 (FOXO1), and apoptotic factors such
as Bax and B cell lymphoma 2 (BCl-2). Cyclin D1 and the
oncoprotein c-Myc are upregulated after the deactivation
of GSK3β. The inhibition of FOXO1 by AKT accelerates
the cell cycle by downregulating cyclin-dependent kinase
inhibitors p27 and p21. The central signaling factor NF-κB
is activated by AKT and is a main signaling factor that
allows cancers to develop and progress and acquire drug
resistance in aggressive malignancies. AKT decreases the
proapoptotic levels of Bad and Bax and conversely
increases the anti-apoptotic levels of Bcl-2, Bcl-xl, and
myeloid cell leukemia 1 (Mcl1). Furthermore, Akt reduces
the release of tumor suppressor p53. The generalized
function of PI3K/AKT signaling pathway is to minimize
apoptosis while increasing cellular proliferation and survival .
Targeting the AKT pathway was demonstrated when
Lu et al. utilized the phytoestrogen
5,7-dihydroxy-4′methoxyisoflavone to induce apoptosis in OS cells. The
phytoestrogen did not have any effect on the normal
human skin fibroblasts but did selectively inhibit the
U2OS cancer cells. The inactivation of the pathway was
confirmed by downregulation of BCL-2 and the
upregulation of the expression of Bax .
H2 relaxin (RLN2) is a peptide hormone and member
of the insulin-like superfamily that has been shown to play
a role in the pathogenesis of OS by positively regulating the
AKT pathway. Ma et al. demonstrated that overexpressing
RLN2 increased OS cellular invasion and migration, while
silencing RLN2 decreased the ability of these cells to invade
and survive. Moreover, the OS cells were more sensitive to
cisplatin chemotherapy when RLN2 was silenced. Western
blot analysis supported the positive direct correlation of
RLN2 and AKT, showing decreased signal intensity of AKT
when RLN2 was inhibited. These results illustrate the
importance of modulating the signaling pathway AKT in the
treatment of OS .
The Ras-Raf-MEK-ERK (along with PI3/AKT) is the
most altered signaling pathway in solid tumor cancers.
The pathway commences with the binding of growth
factors to their receptors which activate the Shc/Grb2/
SOS coupling complex. The complex subsequently
activates the inactive protein Ras which modifies guanosine
diphosphate (GDP) to guanosine triphosphate (GTP). A
downstream target for RAS is RAF kinase. RAF kinase
activates MEK1/2 which will catalyze the activation of
ERK1/2. ERK1/2 in turn can phosphorylate numerous
downstream targets integral in cell differentiation,
proliferation, angiogenesis, and survival  (Fig. 5).
The role of MEK in osteosarcoma invasiveness was
supported when Ye et al. demonstrated that
overexpression of MEK was associated with osteosarcoma growth
and metastasis. IHC showed positive expression of total
protein for AKT, p38 MAPK, IGF-1R, and MEK in
orthotopic mouse primary tumor. However, only
phosphorylated MEK was seen via immunohistochemistry in both
the primary as well as the metastatic tumor. Furthermore,
when the MEK pathway was targeted with the MAPK/
ERK inhibitor U0126, there was a decreased invasive
ability of the OS cells in vitro. Hence, new targeted
therapies could be implicated for the Ras/Raf/MEK/
ERK signaling pathway which potentially could impair
the invasiveness of OS .
Gaining an improved understanding of metastasis involves
identifying associated molecules and pathways that regulate
cell motility and invasion. A family of proteins known as
ERM (ezrin, radixin, and moesin) plays an important role
in linking the actin cytoskeleton and the plasma membrane
of a cell. Ezrin maintains cell motility, promotes cell
invasion, and maintains cell adhesion. While in the dormant
form, ezrin with N-terminal ezrin/radixin/moesin
(ERM)associated domain (N-ERMAD) associates in the cytoplasm
with carboxy-ERMAD (C-ERMAD). Ezrin then becomes
phosphorylated at various sites resulting in transformation
to the active form. The C-terminal of transmembrane
proteins as well as C-ERMAD binds with the N-terminal of
activated ezrin. In addition, ezrin can serve as a linker
protein between specific membranous proteins and F-actin
via ERM-binding phosphoprotein 50 (EBP50). Guanosine
diphosphate inhibitor (GDI) from the Rho-GDI complex is
displaced by activated ezrin. This displacement can then
stimulate PI4P5 kinase activity which is catalyzed by GDP/
GTP exchange factor (GEF). Thereafter, PI4P5 kinase can
act on PIP to convert PIP to phosphatidylinositol
(4,5)bisphosphate (PIP2). Thus, PIP2 sequentially converts
dormant ezrin into the active form  (Fig. 6). Ezrin was
discovered to be integral in OS and metastasis due to its
ability to drive tumor progression by allowing OS
metastatic cells to overcome a variety of stresses. A significant
stress factor is the ability of OS cells to adapt to the new
microenvironment of the secondary metastatic location in
order to survive. The cells must first detach from the
Fig. 6 Ezrin pathway. While in the dormant form, ezrin with N-terminal
ezrin/radixin/moesin (ERM)-associated domain (N-ERMAD) associates in
the cytoplasm with carboxy-ERMAD (C-ERMAD). Ezrin then becomes
phosphorylated at various sites resulting in transformation to the active
form. The C-terminal of transmembrane proteins as well as C-ERMAD
binds with the N-terminal of activated ezrin. In addition, ezrin can serve
as a linker protein between specific membranous proteins and F-actin
via ERM-binding phosphoprotein 50 (EBP50). Guanosine diphosphate
inhibitor (GDI) from the Rho-GDI complex is displaced by activated
ezrin. This displacement can then stimulate PI4P5 kinase activity which
is catalyzed by GDP/GTP exchange factor (GEF). Thereafter, PI4P5 kinase
can act on PIP to convert PIP to phosphatidylinositol (4,5)-bisphosphate
(PIP2). Thus, PIP2 sequentially converts dormant ezrin into the
primary tumor, intravasate into the bloodstream, transport
to the metastatic secondary site, and colonize and
A correlation appears to exist between high levels of ezrin
expression in highly invasive cancer cells and low levels of
ezrin expression in low-invasive cancer cells. In vivo
experiments revealed that when ezrin was knocked down, cell
invasion and migration were reduced. In contrast, when
ezrin was overexpressed, cancer cells had higher ability to
invade and migrate. Interestingly, ezrin expression was
decreased when microRNA-183 (miR-183) was
increased. Ezrin also appeared to show a correlation
with N-cadherin expression. N-cadherin expression is
associated with epithelial-to-mesenchymal transition
(EMT). This particular cadherin is necessary for metastasis
and cancer growth. This mechanism promotes detachment
of the cancer cells from the primary site and facilitates the
migration to blood vessels and secondary sites. Experiments
have validated that ezrin results in increased N-cadherin
Zhang demonstrated that ezrin ectopic overexpression in
the MG63 OS cell line resulted in increased tumor
migration and cell invasion in vitro. Ezrin’s effect on OS was
further demonstrated in vivo through an experimental
metastasis model in which the MG63 OS cells were delivered
to female mice to develop pulmonary metastasis. Ezrin was
notably found to increase N-cadherin and enhance the
expression of the MAPK/ERK signaling pathway. Ectopic
overexpression of ezrin in the OS cell line MG63 promoted
tumor cell invasion and migration. Consistent with this
finding, knockdown of ezrin inhibited tumor cell invasion
and migration. Collectively, these results suggest that
increased N-cadherin and ERK signaling activation by ezrin
can promote aggressiveness in OS .
A meta-analysis was conducted to evaluate the
expression level of ezrin in osteosarcoma patients compared to
patient prognosis. Evaluation of 459 patients revealed
higher frequency of ezrin expression in stage III and
stage IV than in lower histological stages of
osteosarcoma. Positive expression of ezrin correlated with lower
overall survival . Compounds that are able to
successfully inhibit ezrin are being researched to serve as
potential therapeutics for osteosarcoma.
One compound known as NCS305787 was discovered
to directly bind to ezrin , inhibiting its function of
invasive promotion. NSC305787 has a structure very
similar to quinolone-containing compounds such as
antimalarial agents. On the basis that ezrin likely has a key
role in the pathogenesis of malaria, additional
antimalarial compounds were screened to identify novel ezrin
inhibitors with better efficacy and drug properties than
NSC305787. One such compound, MMV667492, had
improved physicochemical properties for drug likeness
compared to NSC305787 and exhibited potent anti-ezrin
activity in biological assays. The drug-like compounds
MMV020549 and MMV666069 also showed promising
activities in functional assays. Both compounds
demonstrated superior activity compared to the NSC305787,
especially in inhibiting pulmonary metastatic growth. These
data demonstrate that anti-malarial compounds warrant
further study in randomized clinical trials of OS .
Osteosarcoma has a predilection for the metaphyseal
regions of the long bones, regions known to represent a
large pool of mesenchymal cells. Several studies have
reported that the stem cells can induce pro-inflammatory
effects through the activation of multiple factors .
This inflammation yields bioactive molecules including
growth factors and cytokines that are able to stimulate
persistent cellular proliferation and subsequent malignant
transformation. Interleukin-6 (IL-6), believed to be one of
the most important inflammatory factors involved in this
inflammatory process, can activate Janus tyrosine kinase
(JAK) family members. These kinase family members
including JAK1, JAK2, and tyrosine kinase 2 (TYK2) can in
turn activate transcription factors of the signal transducer
and activator of the transcription (STAT) family .
The corresponding ligand binds to the cell surface
receptor prompting activated JAK2 protein to phosphorylate
tyrosine residues in the cytoplasmic domain of the receptor.
More so, JAK2 also phosphorylates recruited STAT which
results in STAT dimerization via conserved Src homology 2
(SH2) domains. STAT dimers then translocate to the
nucleus via nucleoprotein interactor 1 (NP-1) where they
induce transcription of target genes. In addition, the JAK/
STAT also interacts with RAS/MAPK, PI3, and AKT
pathways (Fig. 7). Under normal conditions, gene expression is
regulated by negative feedback mechanisms including the
production of the negative regulator suppressors of
cytokine signaling (SOCS) .
IL-6 wields its pro-proliferative effect by binding to the
IL-6 receptor complex. This complex is comprised of
either IL-6R and glycoprotein 130 (gp130) or the soluble
form of IL-6R (sIL-6R). When IL-6 binds to its respective
receptor, the gp130 undergoes a conformation change and
gp130 can then activate signal transducer and activator of
transcription (STAT3). Mounting evidence is showing that
constitutively activated STAT3 in the face of abnormal
dysregulation promotes the development of tumors.
Tu et al. demonstrated that a neutralizing antibody
could block the activation of STAT3 in OS cells  by
way of a compound known as AG490 which is a specific
and potent inhibitor of JAK2. By inhibiting JAK2, STAT3
could not be activated via phosphorylation, and thus,
there was a reduction in the proliferation, migration,
and invasion of OS cells. The effects were also seen in
vivo in a nude mouse model injected with OS cell line
Fig. 7 Jak/STAT pathway. The corresponding cytokine or growth
factor ligand binds to the cell surface receptor prompting activated
JAK2 protein to phosphorylate tyrosine residues in the cytoplasmic
domain of the receptor. More so, JAK2 also phosphorylates recruited
signal transducer and activator of transcription (STAT) which results
in STAT dimerization via conserved Src homology 2 (SH2) domains.
STAT dimers then translocate to the nucleus where they induce
transcription of target genes. In addition, JAK acts as a docking site
for SH2 containing adapter proteins including Src homology 2
domain-containing phosphatase 2 (SHP2), growth factor receptor
bound protein-2 (GRB2), and Src homology 2 domain-containing
transforming protein (SHC). GRB2 which is associated with Son of
Sevenless (SOS) can bind the tyrosine phosphorylated receptor directly
or indirectly by way of the Src homology 2 domain-containing protein
(SHC). This binding results in the translocation of SOS to the membrane.
At the membrane, SOS exchanges GDP for GTP on Ras guanine
nucleotide-binding proteins. Ras-GTP can then activate MAPK cascade.
Aside from RAS, JAK/STAT also interacts with PI3 and AKT pathways .
Under normal conditions gene expression is regulated by negative
feedback mechanisms including the production of the negative
regulator suppressors of cytokine signaling (SOCS)
Saos-2 and then treated with AG490 (Jak2 inhibitor).
There was a significant reduction in tumor growth in
those mice treated with AG490 . Tu et al. further
demonstrated a reduction in pulmonary metastasis and
an overall increased survival in the mice. Thus, AG490
could be a potent inhibitor of OS cells .
Chemopreventive agents and anti-cancer
Curcumin is a naturally occurring compound derived from
the rhizomes of Curcuma longa. A member of the ginger
family, it is a spice that has been commonly used for food
preservation as well as for health care, primarily on the
Indian subcontinent. The compound was first isolated two
centuries ago, and has been used to treat a variety of
systemic diseases, including pulmonary, dermatological,
and gastrointestinal system disorders. Curcumin has been
able to perform many of these functions because it
possesses a wide array of functional characteristics including
antioxidant, antiviral, antifungal, antibacterial,
antiinflammatory, and anti-cancer properties .
The anti-cancer properties of curcumin have been
demonstrated in multiple types of cancers, including
OS. Chang et al. evaluated the cytotoxicity of various
concentrations of curcumin in the OS cell line MG63.
The results demonstrated that the osteoblasts
maintained 80% viability with all the curcumin
concentrations, implying less sensitivity of the osteoblasts to the
curcumin. MG63 had 50% cell viability with 10 μM
curcumin compared to the control osteoblasts,
suggesting increased sensitivity of OS to curcumin. Our own
experimentation has demonstrated that curcumin can
effectively target stem cells in patient-derived OS tumor
samples resulting in reduced osteosphere formation status
post treatment. In addition, live fluorescent staining of
patient-derived OS cells treated with increasing
concentration of curcumin demonstrated propidium iodide staining
the cells’ nuclei representing cell death. These studies
therefore suggest that curcumin can selectively kill
malignant OS cells rather than healthy osteoblasts .
Additional studies have characterized how curcumin
affects OS cancer cells. Li et al. showed that curcumin
inhibited proliferation, activated apoptosis, induced G2/
M phase cell cycle arrest, and decreased the ability of
OS cells to invade and metastasize. These actions were
accomplished by downregulating Notch-1 and the
respective downstream genes, including Hes-1, cyclin D1,
matrix metallopeptidase 2 (MMP-2), and matrix
metallopeptidase 9 (MMP-9) (Fig. 2). This research provided
the first evidence that Notch-1 and its respective
downstream genes are downregulated in response to curcumin
and presented the possibility that curcumin may be an
effective compound for treating OS .
Chang et al. observed that curcumin was able to
induce apoptosis in osteosarcomas by increasing the
reactive oxygen species (ROS) in a
dose-concentrationdependent manner. Furthermore, high concentration doses
of curcumin (80 μM) led to the release of cytochrome C
and activation of caspase-3, prompting apoptosis of MG63
Unpublished data from our lab confirm curcumin’s
dose-dependent inhibitory effects on the proliferation of
OS cells MG63, KHOS, and SJSA. Furthermore,
curcumin in combination with traditional agents including
doxorubicin and cisplatin results in a higher reduction
of OS cells compared to the traditional chemotherapy
alone. Therefore, curcumin appears to have potent
anticancer activity and could be a novel agent to introduce
in upfront therapy.
Numerous approaches are being used to study the effects
of Notch signaling inhibition on many cancer types. These
approaches include use of a neutralizing antibody against
target proteins, use of dominant-negative mutant for key
proteins in Notch signaling, and use of natural synthetic
compounds to target Notch signaling. Inhibition of cancer
progression by natural or synthetic compounds offers
significant promise for reducing cancer incidence and
mortality in patients. Use of diallyl trisulfide (DATS), an
organosulfide derived from garlic, showed inhibition of
proliferation in OS cells by triggering cell cycle arrest and
apoptosis in vitro. DATS also has been reported to
suppress cell survival, wound-healing capacity, invasion, and
angiogenesis in OS cells through decreased expression of
Notch-1 downstream genes, such as vascular endothelial
growth factor (VEGF) and matrix metalloproteinases.
DATS had contrasting effects on various microRNA.
Treatment with DATS decreased tumor promoting
miR21 and increased potential tumor suppressor miR-143 and
miR-145 (Fig. 2). These results suggest that DATS
inhibited osteosarcoma growth and aggressiveness via a
mechanism targeting a Notch-miRNA regulatory circuit .
Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a natural
phenol and phytoalexin produced by several plants in
response to injury or pathogens. Food sources include
mulberries, raspberries, blueberries, grapes, and peanuts
. It was first isolated from roots of the white hellebore
(Veratrum grandiflorum O. Loes). The phenol is an active
constituent of the roots from Polygonum cuspidatum .
Historically, resveratrol has been reported to cause cell
cycle arrest, promote apoptosis, and inhibit cancer cell
proliferation in oral squamous carcinoma, glioblastoma,
liver carcinoma, non-melanoma skin cancers, and thyroid
carcinoma . Rusin et al. reported that resveratrol
inhibited cell growth and induced senescence in OS cells
(U2-OS) by modifying the DNA metabolism. Resveratrol
can alter the localization and expression of critical
proteins integral in cell cycle regulation and DNA repair, as
well as generate instability of the telomeres and promote
DNA damage. Data demonstrated that OS cell growth
was inhibited at 50-μM concentration, and the cells were
arrested in the S phase of the cell cycle (suggesting
interference with the metabolism of DNA) .
Zou et al. reported that resveratrol inhibited the
proliferation of MG63 OS cells by downregulating
βcatenin in the canonical WNT signaling pathway.
Western blot and RT-qPCR determined that the protein and
mRNA expression levels of β-catenin and C-myc were
significantly downregulated (Fig. 3b). Additional
experiments with animal models will need to be performed to
confirm this effect. Nevertheless, this study indicates a
Apigenin (4′,5,7-trihydroxyflavone) is a natural glycoside
that is part of the flavone class. The compound is found
in a multitude of vegetables and fruits such as wheat
sprouts, onions, tea, and oranges . Prior studies have
exhibited that tumor proliferation, invasion, and tumor
growth in prostate cells are inhibited by apigenin .
Moreover, apigenin has demonstrated an inhibitory
effect on pancreatic cancer cell proliferation as well as the
migration and invasion of A2780 human ovarian cancer
cells . Apigenin may serve as therapeutic agent in
the prevention of OS cancers.
The anti-cancer properties of apigenin were
demonstrated with U2OS cells, which underwent apoptotic
induction as well as xenograft tumor growth inhibition.
Apigenin demonstrated a marked effect on impairing
proliferation of OS cells in a time- and dose-dependent
manner in U2OS and MG-63 cells. Impairing cellular
proliferation implied that apigenin was able to inhibit
the survival of both cell lines. The cells were arrested
in the G0/G1 phase after 24 h, indicating that the
decrease in proliferation was in part attributed to cell
cycle arrest. Apigenin was found to also inhibit the
invasion of both U2OS and MG-63 cells. The inhibition
appears to be a result of downregulating the expression
β-catenin in the Wnt signaling pathway. The inhibition
was evident by a reduction in β-catenin (Fig. 3b).
Conversely, overexpression of β-catenin reversed the
inhibiting effect of apigenin .
Cyclopamine is a steroidal alkaloid that has shown
ability to antagonize numerous cancers including breast
cancers, prostate cancers, gastrointestinal cancers, and
OS [31, 58–60]. The compound is derived from the
corn lily (Veratrum californicum) . When
cyclopamine binds to the receptor smoothened (SMO), it
prevents further signal transduction to the target gene
GLI5  (Fig. 4b). This inhibition has demonstrated
interesting results in numerous experimentations dealing
with tumors that are dependent on the Hh signaling
Warzecha et al. demonstrated that treating OS cells with
cyclopamine resulted in a moderate reduction in the
proliferation of the cells, depicted by viability assay. However, the
anti-proliferative effect of cyclopamine was not due to the
compound being a steroidal alkaloid but rather to the
receptor effect on Hh signaling pathway. This notion was
deduced by treating OS cells with another steroidal alkaloid
known as tomatidine. This particular agent in contrast to
cyclopamine lacked receptor activity against Hh signaling
pathway. The tomatidine-treated cells had a proliferation of
51.6% compared to 18.5% from cyclopamine .
Researchers were then able to prove the cytotoxic
effect of cyclopamine on OS cells in vivo. In the
experiments, 5 × 105 Os-50 cells were injected into the tail veins
of young mice. Pulmonary metastasis in the controlled
group of mice was significantly increased compared to the
mice group who were treated with cyclopamine.
Immunohistochemistry with Ki-67 antibody was comparatively
higher in the cyclopamine group compared to the control
group, implying a decreased percentage of
immunoreactive metastatic cells being stained after cyclopamine
Another natural compound that has been investigated
for OS treatment is sulforaphane (SFN). This compound
is a member of the isothiocyanate family and obtained
from cruciferous vegetables including Brussels sprouts,
cabbage, and broccoli . Sawai et al. evaluated the
effect of SFN and radiation treatment on LM8 murine OS
cells. The cells were cultured with multiple
concentrations of SFN that resulted in increased cell populations
in G2/M phase. The combination of SFN and 2 Gy of
radiation suppressed ERK and AKT phosphorylation. It
was also discovered that SFN induced apoptosis through
G2/M phase arrest and inhibited the activation of ERK
and AKT  (Fig. 5). An additional study reported that
SFN contributed to genomic instability in MG63 OS cell
lines as evidenced by an increase in DNA breaks,
nuclear and mitotic abnormalities, and clastogenicity.
Loss of viability was evident by increased formation of
micronuclei and apoptotic bodies . SFN may prove
to be a promising molecular targeting chemotherapeutic
agent for OS cancers .
Osteosarcoma continues to be a challenging cancer to
treat, and there has been a notable lack of progress in
survival statistics for this aggressive bone cancer. Progress
has stalled in part due to the lack of knowledge of OS
pathogenesis. Historically, the lack of understanding of
cellular mediators involved in proliferation and invasion
of OS impaired our ability to target those mediators. As a
result, the same backbone of chemotherapy has continued
to remain the primary treatment strategy. The overall
5year event free survival of pediatric patients with
metastatic OS has been poor at 30% . Simultaneously, there
has been an eruption of scientific research investigating
signaling pathways that appear to play crucial roles in
tumor survival and renewal capacity. Many of these
signaling pathways appear to be susceptible to targeting
with natural compounds. These natural compounds have
the potential to target multiple aberrant pathways in OS.
Numerous in vitro and in vivo studies have demonstrated
that these phytochemicals can modulate the signal
pathways of OS. These various phytochemicals have already
demonstrated considerable efficacy in a variety of other
cancer types. Given the extraordinary lack of progress
seen in OS clinical trials that continue to use various
combinations of cytotoxic chemotherapy, it is time we
look closer at these targeted agents and natural
compounds. We need to quickly elucidate their mechanisms
of action and safety profiles to push them into larger
clinical trials for upfront therapy, so that we can
finally make substantial advancements in treating this
(EBP50): ERM-binding phosphoprotein 50; (NP-1): Nucleoprotein interactor 1;
4E-BP1: 4E-binding protein 1; ADAM: A disintegrin and metalloprotease;
ALDH: Aldehyde dehydrogenase; APH1: Anterior pharynx defective 1; BCl-2: B
cell lymphoma 2; C-ERMAD: Carboxy-ERMAD; CK1α: Casein kinase 1 alpha 1;
DATS: Diallyl trisulfide; Deptor: DEP domain-containing mTOR-interacting
protein; DHh: Desert hedgehog; Dll-1: Delta-like-1; Dll-3: Delta-like-3; Dll-4:
Deltalike-4; DNA-PK: DNA-dependent protein kinase; Dsh/DV1: Dsh/DV1; DSL: Delta/
Serrate/Lag-2; eIF4E: Eukaryotic translation initiation factor 4E; EMT:
Epithelial-tomesenchymal transition; ERM: Ezrin/radixin/moesin; FOXO: Forkhead box O1;
GDI: Guanosine diphosphate inhibitor; GDP: Guanosine diphosphate; GEF: GDP/
GTP exchange factor; GLI1: GLI family zinc finger 1; GLI2: GLI family zinc finger 2;
GLI3: GLI family zinc finger 3; GLI5: GLI family zinc finger 5; GP130: Glycoprotein
130; GSI: γ-Secretase inhibitors; GSK3β: Glycogen synthase 3β; GTP: Guanosine
triphosphate; GβL: LST8/G-protein β-subunit like protein; Hes: Hairy and
enhancer of split; Hey: Hes-related family BHLH transcription factor with YRPW
motif; Hh: Hedgehog; IHh: Indian hedgehog; IL-6: Interleukin-6; ILK:
Integrinlinked kinase; Jag-1: Jagged-1; Jag-2: Jagged-2; JAK: Janus tyrosine kinase;
MAFK: v-maf avian musculoaponeurotic fibrosarcoma oncogene
homolog K; MAML: Mastermind-like proteins; Mcl1: Myeloid cell leukemia
1; MDM2: Mouse double minute 2 homolog; miRNAs: MicroRNAs;
MMP2: Matrix metallopeptidase 2; MMP-9: Matrix metallopeptidase 9;
mSIN1: Mammalian stress-activated protein kinase interacting protein;
mTOR: Mammalian target of rapamycin; N-ERMAD: N-terminal ezrin/
radixin/moesin (ERM) associated domain; NEXT: Notch extracellular
truncation; NF-κB: Nuclear factor-κB; NICD: Notch intracellular domain;
NKD2: Naked cuticle homolog-2 gene; NRR: Negative regulatory region;
PDK1: Phosphoinositide-dependent kinase 1; PEN-2: Presenilin
enhancer2; PIP2: Phosphatidylinositol 4, 5-biphosphate; PIP3: Phosphatidylinositol
3,4,5-triphosphate; PRAS40: Proline rich AKT substrate; PTCH1: Patched 1;
PTCH2: Patched 2; Raptor: Regulatory associated protein of mTOR; RBPJ/
CBF1: Recombination signaling binding protein of hairless-J; Rheb: Ras
homolog enriched in brain; RLN2: H2 relaxin; ROS: Reactive oxygen
species; S6K: S6 kinases; Ser473: Serine 473; SFN: Sulforaphane; Shc: Src
homology 2 domain-containing; SHh: Sonic hedgehog; sIL-6R: Soluble
form of IL-6R; SMO: Smoothened; SOCS: Suppressors of cytokine signaling;
STAT: Signal transducer and activator of the transcription; TACE/ADAM17/
CD156q: Tumor necrosis factor-alpha converting enzyme; Thr308: Threonine
308; Tsc: Tuberous sclerosis complex; TYK2: Tyrosine kinase 2; VEGF: Vascular
endothelial growth factor; β-Trcp: Beta-transducin repeat containing E3
ubiquitin protein ligase
PA and GK drafted the manuscript. DS, KC, KN, PD, and SA discussed and
revised the manuscript. GK designed the figures. PD designed the table. We
thank members of the Anant laboratory for their discussion during the
course of this study. SA is an Eminent Scientist of the Kansas Biosciences
Authority. All authors read and approved the final manuscript.
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