ABSTRACT

The Phosphatidylinositol-3-kinase (PI3K) signaling pathway mediates a number of cellular

processes such as cell growth, proliferation and survival, that are very critical to tumorigenesis and
cancer progression. Due to the critical role of this signaling pathway in various cancer, it is tightly
regulated by a crop of important regulators within the pathway, whose alterations are known to
give rise to various human cancers. In consistency with its role in signaling cellular growth and
proliferation, abnormal activation of the PI3K pathway has been reported by various studies to
play essential role in the initiation and progression of various human cancers. This includes the
widespread germline mutations and somatic gene alterations that leads to the inactivation of the
tumor suppressor gene (PTEN), and the gain of function mutations in the PIK3CA, the gene that
codes for the catalytic subunit of phosphosinositide-3 kinase (p110). The high occurrences of these
mutations in human cancers have led many scientists to evaluate the mechanisms behind the
oncogenic alterations of the PI3K pathway. Consequently, various intracellular components of this
pathway are now targeted in anticancer research/drug discovery, giving rise to current panoply of
clinical use and trials of PI3K, AKT, mTOR, and PDK inhibitors.

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 Table of Content
ABSTRACT 1

TABLE OF CONTENT 2

1.0 INTRODUCTION 3

1.1 MAJOR COMPONENTS OF THE PI3K SIGNALLYING PATHWAY 5

1.1.1 PI3K (Phosphoinositide 3-kinase) 5
1.1.2 PTEN (The Phosphatase And Tension Homology) 6
1.1.3 PDK1 (3 Phosphoinositide-dependent Protein Kinase-1 6

1.2 MAJOR EFFECTORS DOWNSTREAM OF THE PI3K/AKT

SIGNALLYING PATHWAY 7
1.2.1 AKT (Also Known As Protein Kinase B(PKB) 7
1.2.2 mTOR 7
1.2.3 GSK3 8
1.2.4 FOXOs 13
1.2.5 TSC2 10
1.2.6 MDM2 10

1.3 ACTIVATION OF THE PI3K SIGNALLING PATHWAY 10

1.4 PI3K/AKT SIGNALING AND CANCER 12

1.4.1 PI3K/Akt signaling and tumorigenesis 12
1.4.2 PI3K/Akt signaling and tumor growth 12
1.4.3 PI3K/Akt signaling and cancer metabolism 13

1.5 PI3K/AKT SIGNALING AND DRUG RESISTANCE 14

1.5.1 Chemotherapy resistance. 14
1.5.2 Immunotherapeutic resistance 15

1.6 REGULATION OF PI3K SIGNALING PATHWAY 16

1.6.1 Upstream Regulators of PI3K Signaling 16
1.6.2 Feedback Regulators of PI3K Signaling 16

1.7 KNOWN MUTATIONS IN THE PATHWAY 17

1.8 CANCER DRUG DISCOVERY APPROACHES TARGETING

PI3K PATHWAY 18
1.8.1 Inhibitors of the lipid kinase activity of PI3K 19
1.8.2 Inhibitors of the Ser/Thr kinase activity of PDK1 20
1.8.3 Inhibitors of the Ser/Thr kinase activity of AKT 21

1.9 ROLE OF THE PI3K/AKT IN THERAPY RESISTANCE 22

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CONCLUSION 22
REFERENCE 23

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1.0 INTRODUCTION

In recent years, therapeutic approaches to the treatment of cancer have not been very successful,
mainly because of inherent or acquired drug resistance of cancerous cells. Therefore, there is a
need for more research on the underlying biological pathways of carcinogenesis, and to find ways
to target these pathways to design new therapeutic agents that are less prone to cancer drug
resistance. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is one of the common
intracellular pathways whose over activation has led to various cancers in human (Shi et al., 2019).
PI3K pathway regulates a number of cell physiological activities that include cell cycle, growth
and apoptotic cell death. Various studies have noted that the initiation and progression of several
solid tumors are wholly or partly dependent on the deregulation of PI3K signaling pathway.
Therefore, enormous effort has been made in recent times to find new therapeutic agents that target
PI3K signaling, either downstream or upstream of the pathway. (Rascio et al., 2021).

Phosphoinositide 3-kinases (PI3Ks) represents a conserved family of lipid kinases that

phosphorylate the phosphoinositides at the 3’ -hydroxyl group. The most prominent product of
this catalysis is the phosphatidylinositol-3,4,5-trisphosphate (PIP3), an important second
messenger that recruits AKT to activate the signaling of cellular growth, proliferation and survival.
(He et al., 2021). The production of PIP3 is regulated by the Phosphate and Tension Homology
(PTEN), a tumor suppressor which dephosphorylates PIP3 to yield PIP2.

PI3K is divided into three subclasses depending on their affinity for their substrates, and the
specific structural conformations that control various aspects of cell biology. The mostly
researched class of the enzyme is the Class I PI3K, a heterodimer that consists of a regulatory
subunit (p85) and a catalytic subunit (p110). The class I PI3K is further divided into class IA PI3K
and class IB PI3K. Till date, only class IA PI3K has been linked to human cancer, however, the
frequent generic changes associated with the enzyme and its downstream effectors has made PI3K
signaling pathway one the most commonly dysregulated in human cancer. PI3K deregulation has
been reported in various human cancers including breast cancer, hematologic malignancies, and
colorectal tumors, highlighting that targeting the PI3K pathway for cancer drug development could
be potentially useful. To that end, various anticancer drugs in clinical use or preclinical trials are
known to target the PI3K pathway either directly or indirectly.

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1.1 MAJOR COMPONENTS OF THE PI3K SIGNALLYING PATHWAY

1.1.1 PI3K (PHOSPHOINOSITIDE 3-KINASE)

The plasma membrane-associated lipid kinase PI3K is made up of three subunits: the p85
regulatory subunit, the p55 regulatory subunit, and the p110 catalytic subunit. PI3K is split into
three classes based on their different structures and specific substrates: classes I, II, and III. Class
I PI3Ks are divided into two categories: class IA and class IB PI3Ks. The most clearly implicated
in human cancer is Class IA PI3K, which is a heterodimer comprising the p58 regulatory subunit
and the p110 catalytic subunit (Yang et al., 2019). Class IA PI3K contains p110α, p110β and p110δ
catalytic subunits produced from different genes (PIK3CA, PIK3CB and PIK3CD, respectively),
encoded by PIK3CA, PIK3CB, PIK3CG and PIK3CD, respectively. Because of its frequent
mutations, PIK3CA is one of the three class IA catalytic isoforms that is frequently cited in human
cancer. The time it takes for a pathway to become active varies depending on the type of cancer
and the patient. The activation of PIK3CA mutations, for example, is a common early event in
breast and colon cancer. Unlike PIK3CA, transforming mutations in the PIK3CB gene are
uncommon; however, the gene is widely expressed, owing to the isoform's unique method of
interaction with regulatory subunits. PIK3CD is mostly expressed in white blood cells and B cells,
and it is required for the maturation and survival of B cell follicles. Although PIK3CG expression
is linked to cancer growth, it has been shown that lower PIK3CG expression promotes colon cancer
growth and development (Tsolakos et al., 2018).

Class II PI3Ks comprise the C2α, C2β, and C2γ catalytic isoforms and lack regulatory subunits,
allowing them to be activated as monomers Three class II PI3K isoforms have been found in
mammals, with PI3KC2α and PI3KC2β being widely expressed and PI3KC2γ being mostly
expressed in the liver. By altering mitotic spindle formation, PI3KC2α plays an important role in
the progression of breast cancer (Gulluni et al., 2017). Additionally, class II PI3Ks have more
protein-binding domains and a longer N-terminal region, which aids intracellular localization.
Another distinguishing feature of class II PI3Ks is that they cannot produce PIP3 in vitro;
nevertheless, they can generate PIP2 utilizing PIP as a substrate, which differs from class I and III
PI3K functions. Class III PI3K VPS34 (also known as PIK3C3) is unique in that it regulates
autophagy and macrophage phagocytosis by attaching to a protein complex that consists of a
regulatory and catalytic subunit. As a result, heterodimeric class III PI3K can regulate autophagy
as well.

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Figure 1: The domain structure and domain interaction map of the standard regulatory subunit,
p85 and the catalytic subunit, p110 (Acikgoz et al., 2019).

1.1.2 PTEN (THE PHOSPHATASE AND TENSION HOMOLOGY)

The Phosphatase and Tension Homology (PTEN) is a protein and lipid kinase that
dephosphorylates serine, threonine, and tyrosine residues of protein substrates (Wozniak et al.,
2015) PTEN is an important downstream component of the PI3K pathway, acting as a tumor
suppressor to inhibit cellular growth and increase the sensitivity of cells to apoptosis and anoikis
(Porta et al., 2014). PTEN mutations is seen in various advanced human cancers, and its germline
mutation results in Cowden’s disease, a rare hereditary syndrome in human that is associated with
high risks of various cancers that include endometrial, thyroid, and breast cancers. The major lipid
substrate of PTEN is PIP3, which it dephosphorylates to PIP2. Thus, PTEN is acts as a negative
regulator of the PI3K pathway, and its inactivation leads to permanent activation of the PI3K
pathway.

PTEN has two major domains, the C-terminal domain and the N-terminal phosphate domain. The
N-terminal Phosphate domain activity is associated with the majority of its tumor suppression,
however, more than 40% of PTEN mutations that lead to tumorigenesis occur on the C-terminal
domain (Wang et al., 2015). This indicates that the C-terminal domain contains other activities
that are significant in tumor suppressor. The C-terminal domain also contain a tail region in
addition to the C domain, which play important role in the its stability and interactions with other
proteins. The C is also implicated in PTEN stability and its recruitment to phospholipid membranes

1.1.3 PDK1 (3 PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE-1)

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) enzyme is a 556-amino-acid protein

with three distinct motifs: an N-terminal domain, a constitutively activated serine/threonine kinase
domain, and a plecstrin homology (PH) domain at its C-terminus (Di et al., 2017). Unlike PI3K
and AKT, only one isoform of PDK1 has been identified in humans.

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For AKT to be activated fully, it requires phosphorylation at two sites, one within the activation
loop (for example, Thr-308 for AKT1), and one within the C-terminus (for example, Ser-473 for
AKT1). PDK1 phosphorylates the crucial and conserved threonine residue in the activation loop
(commonly referred to as the T-loop) of the three AKT isoforms at the plasma membrane, while
mTORC2 phosphorylates the serine residue. PDK1 phosphorylates and activates other members
of the AGC kinase subfamily, including p70 ribosomal S6 kinase (S6K1), serum and
glucocorticoid-regulated PK, and PKC. (Balasuriya et al., 2018). The known significance of PDK1
in the activation of the above proteins and their downstream effectors suggests that it plays a
significant role in cancer biology in humans.

1.2 MAJOR EFFECTORS DOWNSTREAM OF THE PI3K/AKT SIGNALLYING

PATHWAY

1.2.1 AKT (ALSO KNOWN AS PROTEIN KINASE B (PKB)

AKT dysfunction induces a number of diseases including cancer. In mammals, three isoforms of
AKT including AKT1, AKT2, and AKT3 has been identified. Both AKT1 and AKT2 are richly
distributed in various tissues, while AKT3 is expressing only in the brain cells and its activity is
limited by low distribution (He et al., 2021). Different isoforms of AKT play different role in
cancer, for Instance, AKT2 is associated with cancer cell metastasis, while AKT3 is involved in
hormone independence. The various AKT isoforms also expressed differently in different tissues,
for Instance, AKT2 gene is amplified in pancreatic cancer while AKT1 is overexpressed in various
breast and prostate cancers (He et al., 2021).

AKT is activated by special classes of kinase known as Receptor Tyrosine Kinases (RTK) and G-
protein Coupled Receptors (GPCRs). Once activated, AKT recruits and activate the class I PI3K
isoforms to the plasma membrane. Activate PI3K will further activate the AKT via
phosphorylation of the T ring and the C hydrophobic motifs of AKT. The AKT activated PI3K
also converts PIP3 to PIP2, which in turn activate the Phosphoinositide dependent Kinase 1
(PDK1), a potent activator of AKT (He et al., 2021). Once activated, AKT promotes cell survival,
brother and proliferation through its effect on its wide range of effectors.

1.2.2 mTOR

There have been over 100 Akt substrates found, although not all of them have been verified. The
downstream effects of Akt signaling are extensive due to its parallel regulation of various
substrates. Tumor development, survival, metabolism, and immunity are all said to be regulated
by mTOR. mTOR is an uncommon member of the PI3K-related kinase family as a protein kinase,
and it is frequently organized into complexes, such as mTORC1 and mTOR complex 2
(mTORC2), to play key roles in a variety of biological processes. PI3K activity is at a minimum
under normal circumstances. Signals are sent to PI3K once growth factors are stimulated. The
creation of PIP3, which binds to the PH domain of Akt, is then catalyzed by PI3K. This process is
slowed down by PTEN, a mTOR negative regulator. Akt and mTOR are assumed to work together

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because Akt has been demonstrated to activate mTOR via phosphorylating tuberous sclerosis
complex 2 (TSC2) (He et al., 2021). mTOR, mLST8, raptor, and PRAS40 make up the mTORC1
complex. S6 kinase 1 (S6K1) and eIF-4E-binding protein 1 (4EBP1), both known regulators of
protein synthesis, are phosphorylated by mTORC1. Furthermore, mTORC1- mediated signaling
to HIF1α and LIPIN1 increases glucose metabolism and lipid synthesis, respectively. mTORC2 is
made up mTOR, mLST8, SIN1, and rictor. mTORC2 is formed when activated mTOR interacts
with its protein subunits. When growth hormones activate Akt, mTORC2 phosphorylates it.
Finally, mTOR inhibition has a lot of promise in clinical cancer therapy.

Figure 4: Downstream effectors of the PI3K/Atk signaling pathway and their cellular functions
(He et al., 2021).

The activation of Akt signaling can promote (arrows) or inhibit (blocking arrows) the
phosphorylation of downstream effectors. The phosphorylation of downstream effectors can be
promoted (arrows) or inhibited (blocking arrows) when Akt signaling is activated. Tumor growth,
tumor cell proliferation, tumor survival, cancer immunity, cancer metabolism, and cancer
angiogenesis are all influenced by Akt's downstream regulation. (He et al., 2021).

1.2.3 GSK3

Glycogen Synthase Kinase 3 (GSK3), a multifunctional serine and threonine protein Kinase, was
the first reported downstream effector of AKT. There are two subclasses GSK3, the GSH3α and
GSK3β. Both isoforms share 85% sequence Homology, and were initially identified for their
association with glycogen response to insulin. Different isoforms of GSK3 has specific functions
in different tissues and are known to be expressed at the crossroads of multiple biochemical
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pathways in different diseases. The GSK3 is one of the downstream targets of the PI3K pathway
and it plays important role in tumorigenesis of various cancers. In response to PI3K signaling
induced activation of AKT, GSK3 is activated by the phosphorylation of either Ser21 (α) or Ser9
(β) in N-terminal regulatory domains. This AKT mediated phosphorylation of GSK3 inactivates
the enzyme through an intramolecular pseudo substrate which blocks the GSK3 binding pocket
and prevent the binding of the substrate to GSK3 (Manning et al., 2017). The activity of GSK3
affects a number of biochemical processes on cancer, such as tumor growth and metabolism. GSK3
phosphorylates and inhibits important metabolic enzymes in cancer such its principal substrate,
the glycogen synthase.

1.2.4 FOXOs

Forkhead box Os (FOXOs) are a subgroup of the transcription factor (TF) superfamily that
contains the forkhead box (FOX). FOXO TFs include FOXO1, FOXO3, FOXO4, and FOXO6,
which are all direct downstream targets of Akt. These transcription factors regulate the expression
of many target genes in mammals and are only expressed in certain tissues. FOXO1 and FOXO4
are found mostly in adipose tissue and skeletal muscle, respectively, whereas FOXO3 is found in
the brain, heart, kidney, and spleen. FOXO6, on the other hand, is mostly expressed in the adult
brain, indicating that it plays an essential role in the nervous system. (He et al., 2021The tight
control of FOXO localization in the cytoplasm and nucleus is one of their most distinguishing
features. The nuclear localization signal (NLS) and nuclear export signal (NES) domains of
FOXOs tightly control FOXO shuttling. According to a study, the PI3K/Akt signaling pathway
regulates cell survival partially by phosphorylating FOXOs and increasing their binding to the 14-
3-3 protein, which masks the NLS (blocking nuclear translocation from the cytoplasm but
increasing FOXO removal from the nucleus) and leads to FOXO degradation via the ubiquitin
proteasome pathway (UPP) (He et al., 2021). As a result, the insulin/PI3K/Akt pathway is required
for phosphorylation-dependent adjustment of FOXO levels. Similarly, the extremely conserved
genetic link between Akt and FOXOs supports the concept that Akt and FOXO repression have a
regulatory interaction. This link was discovered in C. elegans, where the loss of FOXO family
members prevented dauer-stage arrest caused by Akt-1 and Akt-2 depletion. The phosphorylation
of threonine 308 and serine 473 regulates Akt activity and substrates. Phosphorylation of serine
473 is not required for Akt-mediated phosphorylation of TSC2 and GSK3, but it is required for
the phosphorylation and deactivation of FOXOs. In conclusion, these biochemical and genetic
studies have demonstrated that FOXO-mediated transcription is responsible for the majority of the
phenotypes generated by Akt depletion; hence, FOXOs are downstream targets of Akt signaling
in a variety of biological processes.

1.2.5 TSC2

The stimulation of growth by PI3K and AKT pathway is induced by growth factors such as IGF1.
The growth factors induced activation of the PI3K pathway leads to the AKT mediated activation
of mTORC1 that leads to the inhibition of tuberous sclerosis 2 (TSC 2). Both TSC1 and TSC2 are

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encoded by a gene is mutated on tuberous sclerosis. The complex of TSC1 and TSC2 inactivates
mTORC1 through the conversion Ras-related Rheb-GTP, a potent activator of mTORC1, to Rheb-
GDP. This process is reversed by the phosphorylation of TSC2.

1.2.6 MDM2

Reduced p53 levels have been linked to cell cycle arrest, while higher p53 levels have been linked
to cell apoptosis. (He et al., 2021). The inhibition of p53 through the activation of another tumor
promoter, MDM2, may cause PI3K/Akt signaling to be activated. MDM2 is a tumor-inducing
oncogene whose mRNA level is regulated by p53 in response to oxidative stress and DNA damage.
(Nakanishi et al., 2014). These findings, combined with the discovery that MDM2 forms a
complex with wild-type p53, show that MDM2 achieves its oncogenic role by interacting with
wild-type p53 and suppressing target gene transcription. PI3K/Akt regulates MDM2's intracellular
location after it has been translated. PI3K/Akt phosphorylates a serine in the MDM2 domain,
according to a study. Because phosphorylation is required for MDM2 to move from the cytoplasm
to the nucleus, active PI3K/Akt can cause MDM2 to translocate to the nucleus, bypassing mitogen
stimulation and activating PI3K/Akt signaling. MDM2 binds to the tumor suppressor p53
following nuclear entrance, inhibiting its transcription and causing its degradation. PTEN
consistently protects p53 from MDM2-induced degradation by suppressing the PI3K/Akt pathway.
Furthermore, p53 expression is linked to DNA damage in a positive way.

1.3 ACTIVATION OF THE PI3K SIGNALLING PATHWAY

For the activation of the PI3K under baseline condition, the regulatory p85 subunit dimerizes with
the catalytic p110 submit to stabilize the structure of PI3K. Under physiological conditions,
extracellular stimuli such as extracellular stimuli, such as growth factors, cytokines, and hormones
are the major activators PI3K. Once activated, PI3K converts PIP2 to PIP3, an important second
messenger that effects pleckstrin-homology (PH), FYVE, Phox (PX), C1, C2 or other lipid-binding
domains of downstream targets to the cell membrane. The PIP3 recruits a number of kinases such
as AKT and PDK, that play important signaling role, to the membrane to activate growth and
proliferation in the cell. This process is regulated by PTEN which dephosphorylates PIP3 to form
PIP2.

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 Figure 2: The overview of PI3K/AKT/mTOR signaling pathway (Yang et al., 2019)

Through genetic screening, AKT has be identified as the primary downstream meditator of the
effects of PI3K activation. When AKT is recruited to the membrane, it undergoes conformational
changes that exposed two ammo acid residues (Thr-308 and Ser-473) necessary for its activation.
Tjr-308 is first phosphorylated by the PDK1, whereas PDK2 phosphorylates ser-473 at the
hydrophobic C-terminal domain to fully activate the enzyme. When AKT is fully activated, its
activity targets a wide range of proteins including GSK3, mTOR, the pro-apoptotic proteins (BAD
and MCM2), cascade9 and a subset of Fork head Transcription factors (FOXO1).

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 Figure 3: The signal transduction downstream of PI3K. (Zhang, 2007)

1.4 PI3K/AKT SIGNALING AND CANCER

1.4.1 PI3K/Akt signaling and tumorigenesis

At present, tumors are generally thought to be propagated by somatic cells, constituting the
tumorigenesis process. Due to dysregulation of their self-detection function, cells are unable to
identify their own mutations and “quit” dividing on time and instead replicate and reproduce with
mutations, resulting in the accumulation of mutations. Moreover, autoimmune deficiencies,
endocrine disorders, and other adverse stimuli also provide conditions that support the process of
tumorigenesis. A study identified AMPK as a vital regulator of Akt activation by various stresses
in tumorigenesis (Han et al., 2018). Other studies have also shown that the PI3K/Akt signaling
pathway regulates its downstream effectors, thereby promoting the occurrence of tumors (Yan et
al., 2019). FOXO is a vital target protein in the PI3K/Akt signaling pathway. After
phosphorylation by PI3K/Akt signaling, the entry of FOXO into the nucleus is blocked, (He et al.,
2021) which prevents the expression of its target genes and eliminates its transcriptional effects on
AR, ERG, Runx2, and other target genes, thereby promoting the occurrence of cancer.

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Based on the important role of the PI3K/Akt signaling pathway in tumorigenesis, some oncogenes
in the PI3K/Akt signaling pathway are positively regulated. For example, KDM5a plays a vital
role in the occurrence of tumors. It promotes the formation of HCC lesions by regulating miR-
433-FXYD3-PI3K-Akt signaling (Ma et al., 2021). Therefore, studies on this pathway and its
related pathways may be crucial to increase the anticancer efficacies of clinical PI3K/Akt
inhibitors.

1.4.2 PI3K/Akt signaling and tumor growth

The uncontrolled proliferation of cancer cells is a key aspect, and the rate of proliferation dictates
the type of tumor therapy. Through signal transduction pathways, EGFRs control tumor cell
growth. The PI3K/Akt signaling system has been found to influence the cell cycle by modifying
its downstream targets, thereby encouraging tumor cell growth. Akt phosphorylates cyclin-
dependent kinase inhibitors and hinders p27 from translocating to the nucleus, reducing its cell
cycle inhibitory function and directly encouraging tumor cell proliferation. Through its
downstream effector p27, Akt also increases tumor cell growth. Because the PI3K/Akt signaling
system has the ability to directly affect tumor cell proliferation, some proteins are implicated in
cancer cell proliferation via the PI3K/Akt pathway. Furthermore, insulin growth factor 2 (IGF2)
affects cancer cell proliferation via modulating the PI3K/Akt pathway, which is implicated in the
development of many cancers (Xu et al., 2018). The PI3K/Akt signaling system is frequently
targeted during cancer treatment to inhibit cancer cell proliferation. For instance, in osteosarcoma
cells, the signaling pathway is controlled by inhibiting lncRNA TDRG1 expression, therefore
interfering with their proliferation (Huang et al., 2020).

1.4.3 PI3K/Akt signaling and cancer metabolism

Metabolic abnormalities, such as alterations in the glycolytic pathway, are one of the most
common characteristics of cancer. Changes in signaling pathways caused by metabolic
abnormalities will affect the occurrence, development, and metastasis of cancer. Insulin, growth
factors, and cytokines activate the PI3K/Akt signaling pathway to regulate metabolism in
organisms under physiological settings. Oncogene activation in the PI3K/Akt signaling pathway
reprograms cellular metabolism in cancer cells by increasing the activities of nutrient transporters
and metabolic enzymes, hence supporting the anabolic demands of abnormally growing cells.
(Hoxhaj and Manning, 2020).

PI3K/Akt signaling does not only modulates metabolism-related proteins like SREBP and changes
metabolism through phosphorylation mediated by metabolic enzymes, but it also affects
metabolism indirectly by influencing different transcription factors (He et al., 2021). Acute
changes in the activity of metabolic pathways and the directionality of metabolic fluxes are caused
by phosphorylation of metabolic enzymes, but long-term alterations in cellular metabolism are

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mainly done through altering gene expression programs (Hoxhaj and Manning, 2020Akt regulates
cellular metabolism through activating mTORC1, GSK3, and FOXO, in addition to directly
phosphorylating a range of metabolic enzymes and nutrient transport regulators (Manning and
Toker, 2017). TSC2 activation by Akt causes Rheb-GTP aggregation, which activates mTORC1
and improves glucose metabolism and lipid synthesis (He et al., 2021). GSK3, a critical regulator
of cellular metabolism, engages in cellular metabolism by phosphorylating and inhibiting
metabolic enzymes like glycogen synthase, one of its substrates (He et al., 2021). FOXO
transcription factors control cellular metabolism and tumor suppression. FOXO is phosphorylated
by Akt, which stops it from accessing the nucleus and regulating the expression of its target genes.
Furthermore, accumulating evidence suggests that PI(3,4)P2 is not simply a waste product of
PI(3,4,5)P3 elimination, but also a signaling molecule in cancer metabolism (He et al., 2021).

1.5 PI3K/AKT SIGNALING AND DRUG RESISTANCE

In tumor therapy, cancer drug resistance is the main reason for treatment failure and indirectly
promotes tumor progression. The dysregulation of PI3K/Akt signaling also plays an important role
in cancer drug resistance. Similarly, a study showed that targeting PI3K/Akt signaling pathway
components can be used to overcome drug resistance in cancer therapy (He et al.,2021).

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 Figure 4: Various targets of PI3K pathway in Growth, survival, translation and
proliferation. (Lee et al., 2014)

1.5.1 Chemotherapy resistance.

Chemotherapies are mainly used to destroy tumor cell DNA and thereby prevent the cells from
replicating, eventually affecting cell survival. During routine chemotherapy, no treatment interval
exists, allowing resistant cells to be generated and leading to tumor regeneration. In addition, DNA
destruction is prevented in drug-resistant cells due to their dormancy (Vives et al., 2013). The
PI3K/Akt signaling pathway is important for the drug resistance of different types of cancer, such
as lung cancer and esophageal cancer. PI3K/Akt inhibitors inhibit tumor growth and induce tumor
cell apoptosis. For NSCLC cells with high Akt expression, the use of PI3K/Akt signaling pathway
inhibitors increases their cell apoptosis induced by chemotherapy and reduces their resistance to
chemotherapy. Furthermore, inhibition of the PI3K/Akt signaling pathway effectively improves
drug induced lung cancer cell apoptosis. (He et al., 2021). Moreover, members of the PI3K/Akt
signaling pathway play an important role in antiestrogen resistance in breast cancer.

1.5.2 Immunotherapeutic resistance

The discovery and application of immune checkpoint inhibitors have substantially advanced the
treatment of malignant tumors. Thus far, CTLA-4, PD-1 and PD-L1 have achieved significant
clinical efficacy and have been approved for the treatment of many types of cancer (Syn et al.,
2017). However, the problem of immune drug resistance persists. Some patients do not respond to
immunotherapy, while other relapse after immunotherapeutic treatment. The PI3K/Akt signaling
pathway plays an important role in the regulation of immune checkpoints and sensitivity to
immune checkpoint inhibitors. Studies have shown that the activation of the Akt signaling pathway
caused by the deletion of PI3KCA mutations is strongly related to the upregulation of PD-L1
expression in the prostate gland (He et al., 2021). In mouse lung cancer models and human lung
cancer cell lines, the Akt signaling pathway regulates the expression of PD-L1 at the protein
translation level (Lastwika et al., 2016) In conclusion, the PI3K/Akt signaling pathway is
obviously related to immune resistance, which provides a basis for combined therapeutic strategies
including immune checkpoint inhibitors and PI3K pathway inhibitors in the future

1.6 REGULATION OF PI3K SIGNALING PATHWAY

1.6.1 Upstream Regulators of PI3K Signaling

As noted earlier, PI3K activation is mainly upon nutrient and growth factor stimulation. The p85
regulatory subunit binds to receptor tyrosine kinases, to activate PI3K (Rathinaswamy and Burke,
2020) In a prototypical example, the binding of the enzyme by the tyrosine kinase receptors will
lead to the binding of the p85–PI3K1A heterodimer to mediate the resultant signal transmission
from the extracellular molecule to the intracellular region. PI3K can also be activated by RTKs

15
through their engagement of Ras and the resulting binding of the GTPase to the Ras-binding
domain (RBD) of PI3K (Hakuno and Takahashi, 2018).

The extent of PI3K activation is regulated negatively through a number of proteins. PTEN is the
principal negative regulator through dephosphorylation of the phosphoinositides in position 3 to
reverse the effect of PI3K. At the transcriptional level, the cellular concentration of PI3K is
controled by the regulation of the transcription factor NF-kB, PPARb/d agonists and TNFa (Kim
et al., 2014) to repress the expression of PTEN. This is particularly importance in lung cancer cells
where downregulation of PTEN by PPARs promotes cancer cell proliferation. On the other hand,
ubiquitinylation-dependent proteasomal degradation of PTEN reduces its levels. NEDD4-1 is the
only identified E3 ligase for PTEN. However, PI3K positively regulate the NEDD4-1 transcription,
therefore representing positive feedback for PTEN degradation and PI3K activation.

Promyelocytic leukemia (PML), a well characterized tumor suppressor protein has be uncovered
recently as a key regulator of the PI3K at multiple levels. PML was found to regulate PP2A activity
toward nuclear AKT residing in the nuclear bodies.

1.6.2 Feedback Regulators of PI3K Signalling

The most prominent negative feedback regulating PI3K pathway is the one triggered downstream
of mTORC1. The first observation of the ability of PI3K downstream components to regulate PI3K
activation arose from a series of studies showing that chronic insulin stimulation led to the
phosphorylation and proteasomal degradation of the adaptor protein IRS-1 (Haruta et al., 2015).
The mechanism of this transcriptional repression depends on the activation of mTORC1 signaling,
as rapamycin treatment and silencing of S6K genes restores IRS-1 levels.

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 Figure 5: Feedback regulation of PI3K pathway. (Carracedo Pandolfi, 2008)

Loss of function of the TSC complex has also been shown to impact RTK levels. It has been
demonstrated that mTORC1 hyperactivation by either Tsc2 or PTEN loss, as well as by PI3K
activation, leads to the repression of PDGFR A and B transcription, which impacts not only in
PDGF signaling to AKT but also on the proper transmission of the signal from other growth factor
receptors. Overall, these data provide support for the concept that this negative regulation of PI3K
by downstream components may be of great importance in cell physiology, as well as in
pathological conditions.

Figure 6: PI3K in tumor microenvironment, playing an important role in tumor initiation,

growth and proliferation. (Yuan and Cantley, 2008)

1.7 KNOWN MUTATIONS IN THE PATHWAY

The PI3K pathway is unique, in that all of the major elements of this pathway have been found
mutated or amplified in a broad range of cancers. Numerous tumor types are thus likely to benefit
from the development of PI3K inhibitors. The axis of PI3K signaling in cancer begins with
engagement of growth factors by RTKs. PI3K is then recruited to plasma membrane anchored
receptors and is activated and phosphorylates PIP2 to generate PIP3. Through its pleckstrin
homology (PH) domain, the nodal kinase AKT (also known as PKB) binds to PIP3, where it is
activated by two phosphorylation events, and triggers a complex cascade of signals that regulate
growth, proliferation, survival and motility. The lipid phosphatase, PTEN, antagonizes this process
by dephosphorylating PIP3 to inhibit activation of AKT. Unlike any other major signaling pathway
in the cell, every member of this signaling axis is frequently altered in cancer.

The PI3K pathway is unique, in that all of the major elements of this pathway have been found
mutated or amplified in a broad range of cancers. Numerous tumor types are thus likely to benefit
from the development of PI3K inhibitors. The axis of PI3K signaling in cancer begins with

17
engagement of growth factors by RTKs. PI3K is then recruited to plasma membrane-anchored
receptors and is activated and phosphorylates PIP2 to generate PIP3. Through its pleckstrin
homology (PH) domain, the nodal kinase AKT (also known as PKB) binds to PIP3, where it is
activated by two phosphorylation events, and triggers a complex cascade of signals that regulate
growth, proliferation, survival and motility. The lipid phosphatase, PTEN, antagonizes this process
by dephosphorylating PIP3 to inhibit activation of AKT. Unlike any other major signaling pathway
in the cell, every member of this signaling axis is frequently altered in cancer

Figure 7: Some frequently mutated components in the PI3K signaling axis.

(Yuan and Cantley, 2008).

1.8 CANCER DRUG DISCOVERY APPROACHES TARGETING PI3K PATHWAY

Blocking the PI3K/AKT pathway could therefore simultaneously inhibit the proliferation and
growth of tumor cells and sensitize them toward programmed cell death. Thus, drug discovery
activities have been directed over the past few years to identify compounds that effectively and
specifically disrupt this pathway in tumor cells.

18
Figure 11: Some targets in PI3K pathway for development of cancer drugs. (Lee et al., 2014)

1.8.1 Inhibitors of the lipid kinase activity of PI3K

Like the majority of protein kinase inhibitors, all existing PI3K inhibitors bind competitively in
the ATP-binding pocket of the catalytic domain. This strategy has enabled the development of
both panPI3K-and isoform-specific inhibitors. Loosely discriminate inhibitors that target multiple
PI3K isoforms may more thoroughly shut down PI3K signaling for the treatment of acute life-
threatening diseases (Yang et al.,2019). The potential toxic side effects on glucose metabolism
and the immune response may be tolerated with short-term use of pan-specific inhibitors.

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 Wortmannin

Figure 12: Representative example of inhibitors of PI3K.

The discovery of Wortmannin and LY294002 as competitive ATP binders revealed what we now
know to be the vast potential of targeting the ATP-binding site of p110. Wortmannin is a potent
pan-specific inhibitor that occupies the ATP-binding site of p110 by forming a covalent bond
between C20 of the wortmannin furan ring and K802 of p110a, disrupting a conserved lysine
residue that is involved in phosphate binding reaction. However, wortmannin has a half-life of
only a few minutes in serum due to the highly reactive C20 position. Wortmannin derivatives such
as PX-866 have been shown to be significantly more stable in vivo, with cytostatic effects when
used as monotherapy. A stabilized wortmannin prodrug also shows promise for treating cancers.

LY294002 (Lilly, Indianapolis, IN, USA), a reversible synthetic compound, makes a key hydrogen
bond between the morpholino oxygen in the compound and the backbone amide of V882 of p110g,
mimicking the interaction made by the adenine of ATP. Variations in this key interaction are
conserved in all existing PI3K inhibitors. SF1126 (Semafore, Indianapolis, IN, USA) is a
LY294002 prodrug that utilizes an RGDS peptide to increase plasma half-life and target drug
delivery to the tumor vasculature. PI-103 (Piramed, Slough, USA) and ZSTK474 (Zenyaku
Kogyo, Tokyo, Japan) share LY294002’s arylmorpholine structure and are potent pan-PI3K
inhibitors. ZSTK474 inhibits all class I PI3Ks (IC50=16–49nM) and has antitumorigenic effects on
a wide variety of xenografts. PI-103 preferentially inhibits p110a (IC50=11nm) as well as
mTORC1/2 (IC50=2/83nM, respectively), and in combination with erlotinib or radiation therapy
has been effective in the treatment of xenografts of glioblastoma cell lines (Chen et al.,2018).

1.8.2 Inhibitors of the Ser/Thr kinase activity of PDK1

UCN-01

Figure 13: Representative example of inhibitors of PDK1

One of the most potent, but nonselective PDK1 kinase inhibitor reported to date is UCN-01
(IC50=6–33nM). This staurosporine-based compound, isolated from the culture broth of
Streptomyces sp, was originally developed as an inhibitor of calcium-dependent PKC. UCN-01

20
inhibits a broad array of PKs, including other members of the AGC subfamily of enzymes (for
example, IC50=491nM for AKT). UCN-01 has been tested in advanced cancer patients in phase
I/II clinical trials, both as single agent and in combination with conventional chemotherapeutic
drugs (for example, carboplatin, fludarabine or topotecan); however, significant antitumor
activities have not yet been reported (Signore et al., 2014)

A class of aminopyrimidine derivates has also been reported to inhibit PDK1 kinase activity. A
representative example of this compound class is BX-320, an ATP-competitive PDK1 inhibitor
(IC50=39nM) that displays good selectivity over AGC family members (for example, 35-and 146-
fold for PKA and PKC, respectively) and other PKs (Rettenmaier et al., 2014). BX-320 blocks the
growth in soft agar of a wide range of tumor cell lines (IC50=0.093– 1.32mM), including PTEN-
negative tumor cell lines such as U87MG and PC3M. The compound is also efficacious in a
metastasis mouse model—LOX melanoma tumors—when administered orally at 200 mg/kg b.i.d.
The resolution of the X-ray structure of the kinase domain of PDK1 with BX-320 revealed a
classical two H-bond interactions between the hinge region—the amino-acid backbone of Ala-
162—of the enzyme and two of the aminopyrimidine nitrogens of the inhibitor. In this context, the
mode of binding of BX-320 is similar to the one reported for aminopyridines in complex with
cyclin-dependent kinase 2/cyclin A. The BX series of molecules has not yet entered into the
clinical trials at the time of this review.

More recently, the identification and initial medicinal chemistry optimization of an indoline-based
series of PDK1 modulators has been reported (Islam et al., 2007). Using a homology model of the
initial indolinone hit bound to PDK1, BX-517 was identified as a potent PDK1 inhibitor
(IC50=6nM) with significant selectivity over PKA (IC50=1600nM; Islam et al., 2007). Although
not fully understood, the observed selectivity over PKA was rationalized on the basis of negative,
steric interactions between the urea moiety of the compound and the side chain of the gatekeeper
residue, methionine, of PKA.

1.8.3 Inhibitors of the Ser/Thr kinase activity of AKT

Nonselective AKT inhibitors have been extensively used as tool compounds to elucidate the role
of this kinase in the biology of human cancers. AKT isoforms are potently inhibited by
promiscuous kinase inhibitors like staurosporine (IC50=48–11nM for AKT1) and derivatives
thereof Similarly, Ro-31-8220 is a potent, but nonselective pan-AKT inhibitor, with primary
activity against PKC (IC50=10nM) and more modest activity against AKT1 (IC50=240nM)
(Nitulescu et al., 2016).

21
 Staurosporine Ro-318220 A-443654

Figure 14: Representative examples of inhibitors of AKT

Promising AKT inhibitors have also been obtained from indazole–pyridine-based derivatives
Exemplified by A-443654 (Ki=160pM for AKT1), these compounds are reversible, ATP
competitive inhibitors, which decrease the phosphorylation of AKT downstream targets in cells
((Nitulescu et al., 2016). (for example, GSK3a/b, FOXO3, TSC2 and mTOR) and in vivo in a
dose-dependent manner Interestingly, these biological effects appear to induce AKT Ser-473
phosphorylation in human cancer cell lines, including PTEN and TSC2-deficient cell lines (Han et
al., 2018). Although several possibilities were considered (for example, a conformational change
in the structure of the compound-bound AKT that prevents Ser-473 dephosphorylation or changes
in AKT relocalization).

1.9 ROLE OF THE PI3K/AKT IN THERAPY RESISTANCE

Inhibition of components of the PI3K pathway can synergize with, or overcome resistance to,
chemotherapy, radiation therapy, hormone therapy and targeted agents in cancer. Potential
mechanisms for this synergy include potentiation of apoptosis (Luszczak et al., 2020). However,
most of these studies have been performed with relatively nonspecific compounds and could
reflect inhibition of PI3K-like kinases activated by DNA damage/radiation (for example, DNA-
activated protein kinase (DNA-PK), ATM and ATR) by drugs such as LY294002 or wortmannin.
In breast cancer, there are important interactions between the genomic and non-genomic effects
of the oestrogen receptor and cytoplasmic/membrane kinases, including members of the PI3K
pathway that play a role in anti-oestrogen resistance. PTEN activity contributes to the efficacy
of trastuzumab (Herceptin; Genentech) and radiation therapy. The activation state of the PI3K
pathway therefore contributes to tumor resistance to targeted therapeutics as well as to
chemo/radiation therapy (Wang and Xu, 2019)

2.0 CONCLUSION

The PI3K signaling pathway play an indispensable role in cell growth and survival, these roles
made it a frequent target for oncogenic transformations. The pathway has increased in complexity
since discovery. In consistency with the critical role of PI3K signaling pathway in cell growth and
survival, it is a frequent target for oncogenic transformation. Mutations in gene encoding a number
of proteins in the pathway are frequently found in various human cancers. Therefore,

22
understanding of the biochemical roles and genetic background of the different components of the
pathway is essential to determine their precise operational circuit, understand their physiological
role, and design effective therapeutic interventions in various cancer.

23
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