**2. The PI3K/AKT/mTOR pathway**

The PI3K/AKT/mTOR constitutes an important pathway downstream of growth factor tyrosine kinase receptors, thus regulating a plethora of biological processes as angiogenesis, proliferation, metabolism, survival, and differentiation [3]. Accumulating evidences indicate,

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therefore, that alterations in the PI3K/AKT/mTOR axis play critical and multifaceted role in cancer pathogenesis and progression. Indeed, systematic analysis performed in 3.281 tumors from 12 cancer types of the Cancer Genome Atlas Pan-Cancer effort has revealed that elements of the PI3K/AKT/mTOR signaling pathway are among the highest frequently mutated genes in cancer, such as uterine corpus endometrioid, breast, colon, lung, head and neck, and ovarian carcinomas [4,5].


**Table 1.** The PI3K proteins family

PI3K is a heterodimer of its catalytic and regulatory subunits and has been classified as class I, II, and III. Class I PI3K is constituted by four 110-kDa catalytic subunits and two main regulatory domains, which is subdivide in class IA and IB. Class IA PI3K (PI3K α, β, and δ) is activated by receptors with tyrosine kinase activity, and class IB PI3K (PI3K γ) is activated by G protein-coupled receptors. The class IA enzymes are dimers of p110α, p110β, or p110δ catalytic subunits and the regulatory subunits p85α (or its splice variants p55a and p50a), p85β, p55γ, p101, or p84 [6,7]. In turn, class IB enzymes are dimers of p110γ catalytic subunit and either p101 or p84 (also known as p87PIKAP) regulatory subunits [8]. The four class I catalytic isoforms share overlapping but distinct functions. Although the expression of p110c and p110d isoforms seems to be confined to immune cells, p110a and p110b are ubiquitously expressed but exhibit isoform-specific cell-type- and context-dependent requirements, thus being involved in a wide range of cellular effects [9–13]. Class II PI3K (PI3KC2) subfamily has additional domains in both N- and C-terminal extensions and exists as 3 isoforms, PI3K-C2α, PI3K-C2β, and PI3K-Cγ [14]. On the other hand, class III PI3K occurs as a single isoform constituted by the catalytic subunit Vps34p and regulatory subunit Vps15 [14] (Table 1).

therefore, that alterations in the PI3K/AKT/mTOR axis play critical and multifaceted role in cancer pathogenesis and progression. Indeed, systematic analysis performed in 3.281 tumors from 12 cancer types of the Cancer Genome Atlas Pan-Cancer effort has revealed that elements of the PI3K/AKT/mTOR signaling pathway are among the highest frequently mutated genes in cancer, such as uterine corpus endometrioid, breast, colon, lung, head and neck, and ovarian

> p110β PIK3CB p110δ PIK3CD

p85β PIK3R2 p55γ PIK3R3

p84, p87 PIK3R6

PI3KC2β PIK3C2B PI3KC2γ PIK3C2G

PI3K is a heterodimer of its catalytic and regulatory subunits and has been classified as class I, II, and III. Class I PI3K is constituted by four 110-kDa catalytic subunits and two main regulatory domains, which is subdivide in class IA and IB. Class IA PI3K (PI3K α, β, and δ) is activated by receptors with tyrosine kinase activity, and class IB PI3K (PI3K γ) is activated by G protein-coupled receptors. The class IA enzymes are dimers of p110α, p110β, or p110δ catalytic subunits and the regulatory subunits p85α (or its splice variants p55a and p50a), p85β, p55γ, p101, or p84 [6,7]. In turn, class IB enzymes are dimers of p110γ catalytic subunit and either p101 or p84 (also known as p87PIKAP) regulatory subunits [8]. The four class I

**Subunit Protein Gene name (human)**

Catalytic p110α PIK3CA

Regulatory p50α, p55α, p85α PIK3R1

Catalytic p110γ PIK3CG Regulatory p101 PIK3R5

Catalytic PI3KC2α PIK3C2A

Catalytic Vps34 PIK3C3 Regulatory Vps15 PIK3R4

carcinomas [4,5].

130 Updates on Cancer Treatment

Class I Class IA

Class IB

Class II

Class III

**Table 1.** The PI3K proteins family

The PI3K family recruits effector proteins, altering their localization, activity, and conforma‐ tion. There are some binding proteins domains that mediate such events [14]. The bestcharacterized domains among them are FYVE (Fab 1, YOTB, Vac 1, EEA1) [15–17], PH (pleckstrin homology) [18], and PX (Phox) [19-23]. Nonetheless, the peculiar composition of the three PI3K subfamilies results in the activation of distinct cellular functions.

In brief, after activation by receptor tyrosine kinases, including members of platelet-derived growth factor receptor, the insulin and insulin-like growth factor 1 (IGF-1) receptors and human epidermal growth factor receptor family (EGFR and HER2), PI3K phosphorylates phosphatidylinositol 4,5-trisphosphate (PIP2) to generate phosphatidylinositol 3,4,5-trisphos‐ phate (PIP3) [24]. In physiological conditions, the level of PIP3 is strictly regulated by PTEN (phosphatase and tensin homolog), a phosphatase that specifically catalyzes the dephosphor‐ ylation of PIP3, converting PIP3 back to PIP2, thus constituting an important endogenousnegative feedback loop of the PI3K signaling pathway [25,26]. The lipid product of PI3K, PIP3, recruits a subset of signaling proteins with PH domains to the membrane, including 3 phosphoinositide-dependent protein kinase (PDK1) and AKT, resulting in its phosphorylation at threonine-308 and activation [24].

In both physiological and pathological conditions, AKT exists in three isoforms in mammals: AKT1, AKT 2, and AKT 3 [27,28]. AKT phosphorylates tuberous sclerosis complex 2 (TSC2), thereby inhibiting the GTPase activity of the TSC1/TSC2 complex and enabling mTOR activation by RAS homologue enriched in brain (RHEB), thus allowing signal propagation [26,29]. mTOR exists in two different structural protein complex: mTORC1 and mTORC2, each of which is expressed in different subcellular compartments, therefore affecting their activation and function. mTORC1 complex is composed of a catalytic subunit mTOR, regulatoryassociated protein of mTOR (RAPTOR), mammalian lethal with SEC13 protein 8 (MLST8), and the noncore components PRAS40 and DEP domain-containing mTOR-interacting protein (DEPTOR). Once activated, mTORC1 leads to increased protein synthesis via its effectors, named translation-regulating factors ribosomal S6 kinase-1 (S6K-1) and eukaryote translation initiation factor 4E binding protein-1 (4EBP-1). S6K-1 and 4EBP1 are major regulators of protein translation [30]. On the other hand, mTORC2 is composed by rapamycin-insensitive compan‐ ion of mTOR (RICTOR), MLST8, and mammalian stress-activated protein kinase interacting protein 1 (SIN1). The function of mTORC2 remains not fully understood, but it is required to phosphorylate AKT at serine-473, thus resulting in its maximal activation [31]. Of clinical relevance, differently from mTORC1, mTORC2 is insensitive to rapamycin inhibition, opening an avenue for drug discovery in face of the development of resistance by cancer cells against first-generation mTOR inhibitors (rapalogs) that particularly target mTORC1 [32] (Figure 1).

**Figure 1.** Overview of Pl3K/AKT/mTOR signaling pathway and some inhibitors of this pathway in clinical studies. The activation of the PI3K by receptor tyrosine kinases promotes conversion of PIP2 to PIP3. PTEN dephosphorylates PIP3, negatively regulating the PI3K signaling. The phosphorylation and activation of AKT impacts many downstream ef‐ fectors, such as mTORCI, and finally leads to multiple cellular processes.

#### **3. The role of PI3K/AKT/mTOR in cancer**

Somatic mutations and/or gains and losses of genes are possible genetic alterations affecting the PI3K/AKT/mTOR pathway in different solid and hematological tumors [33,34]. Indeed, PI3K pathway can be activated by direct upstream signs and can be intrinsically activated due to gain of functional mutations or amplifications in PIK3CA (p110 subunit), mutations in PIK3R (p85 subunit), and mutations or amplifications in one of the AKT isoforms or loss of PTEN [35]. Loss of PTEN via inactivating mutations, due to either copy number loss or homozygous deletions, is associated with both resistance to chemotherapy and reduced survival of human patients [3].

PIK3CA mutations in primary breast tumors have been associated with lymph node metasta‐ ses and overexpression of ER, PR, and HER2 [36]. Furthermore, the presence of activating PI3KCA mutations and loss of PTEN in HER2-overexpressing cancers is correlated with a lower response to trastuzumab and lapatinib [37]. In non-small cell lung cancer, the downre‐ gulation of PTEN is also related with poor prognosis [38,39]. In ovarian cancer, PI3K/AKT/ mTOR molecular alteration appears to be histological subtype specific. Studies have described amplifications in PIK3CA, amplifications of one of the AKT isoforms, and PTEN deletions in 20%, 15%, and 5% of the high grade serous ovarian cancer (HGSOC) cases, respectively [40,41]. The individual mutations, rare events in HGSOC, are prevalent in low grade serous, mucinous, endometrioid, and clear cell ovarian cancer; 20% of endometrioid and 35% of clear cell ovarian tumors display these PIK3CA mutations [42,43]. Besides, copy number changes in the genes encoding PIK3CA and PIK3CB subunits have been associated with a poor prognosis, and the inhibition of PI3K/mTOR was found to delay tumor growth and prolong survival [44,45].

Moreover, mutations of mTOR itself and/or in components of mTOR-related signaling pathways have frequently been described in human malignant diseases [46-48]. Different genetic lesions that mediate mTORC1 activation have diverse consequences: PTEN loss uncouples mTORC1 activation from growth factor signaling; liver kinase B1/serine/ threonine kinase 11 (LKB1/STK11) mutations allow mTORC1 activation despite nutrient deprivation in poorly vascularized tumors; P53 mutations uncouple DNA damage from the inhibition of bioenergetic processes and cell cycle arrest [49]; and hyperactivation of S6K-1, 4EBP1 and eIF4E, and cancer growth by activating the lipid and protein biosynthesis. Furthermore, the increased phosphorylation of mTOR is associated with acquired cispla‐ tin resistance, and AKT signaling has been implicated in primary platinum resistance [50]. In fact, AKT or mTOR inhibitors likely restore chemosensitivity to platinum derivates *in vitro* and in xenograft models [51,52].

These molecular alterations, in addition to the druggability of the components of the PI3K/AKT/mTOR signaling cascade, suggest that targeting the pathway might represent a useful treatment strategy in the fight against cancer.
