Recently, several review papers have provided comprehensive summaries of the biological and biochemical activities of three HPV oncoproteins: E6, E7 and E5 [
86-
88]. Here, we focus on discussing the oncogenic activities of HPV E6, E7 and E5 proteins in inducing the PI3K/Akt/mTOR signalling pathway (Figure
1). Human keratinocytes, a special type of epithelial cells that have a finite life span and do not undergo spontaneous immortalization, are the host cells of HPV infection, [
89]. Following HPV infection, the keratinocytes are immortalized and transformed by the viral oncogenes (E6/E7) that act on multiple cellular events including inhibition of p53 and pRb [
90-
92], altered expression of multiple genes (approximately 4% of the genes on the array) [
26] and activation of several signalling pathways, especially, the PI3K/Akt/mTOR signalling pathway [
89,
93-
95]. The PI3K/Akt/mTOR pathway may in turn mediate multiple cellular functions necessary for HPV-induced carcinogenesis (Figure
1) [
96-
98].
E6 oncogene
HPV E6 oncoproteins are the key players in HPV-induced cancers. The E6 oncoproteins from high-risk mucosotrophic HPVs (α-HPVs) target not only P53, but also a range of host-cell proteins for proteasome-mediated degradation, resulting in alteration of multiple cellular and molecular events [
99-
101]. A genome-wide analysis has shown that E6 up-regulates many genes at the transcript level associated with cancer hallmarks including cell cycle, migration, PI3K/Akt /mTOR signalling to mediate cellular transformation [
102]. The high-risk HPV E6 oncoproteins contain a PDZ-binding domain; a common structural domain of 80–90 amino acids found in the signalling proteins of multiple organisms [
103]. The PDZ-binding domain plays a key role in HPV-mediated cellular transformation. Through this domain, the E6 targets a member of the group of PDZ domain-containing molecules that are mediated by the PI3K/Akt signals [
98,
102,
104]. For example, HPV 16/18 E6 proteins promote proteasome-mediated degradation of human disc large (hDlg) tumor suppressor protein by binding to the second PDZ domain of the hDlg through their C-terminal xS/TxV/L (where x represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif [
2,
105]. High-risk HPV E6 oncoproteins efficiently degrade members of the PDZ domain-containing membrane-associated guanylate kinase (MAGUK) family and a PDZ protein, Na (+)/H (+) exchange regulatory factor 1 (NHERF-1) [
103]. E6 degrades MAGUK by binding to it with inverted domain structure 1 (MAGI-1), which is one of the most strongly bound PDZ domain-containing substrates of E6. E6 interacts with MAGI-1 to facilitate the perturbation of tight junctions. Restoration of MAGI-1 expression in HPV positive tumour cells induces cell growth arrest and apoptosis [
106].
HPV E6 variants (E6*) can act as an adaptor molecule linking a ubiquitin ligase to target proteins, which contain class 1 PDZ domains and are involved in cell junction stability and signalling [
100]. E6* proteins differentially modulate hDlg degradation to rebound the levels of activated PTEN and Akt and strongly enhance expression of p-PI3K contributing to activate MAPKs and promote cell proliferation [
2,
102]. High-risk HPV E6 can target certain substrates both directly and indirectly through the E6* proteins and the two E6 proteins may cooperate in their degradation [
100]. In the absence of full-length HPV-18 E6, HPV-18 E6* expression also downregulates the expression levels of Akt, Dlg, and Scribble [
100]. It has also been reported that HPV16 E6 and HPV18 E6* oncoproteins activate MAPK signalling pathway to promote cell proliferation by upregulating p-PI3K [
102,
107]. HPV18 intra-type variations may result in differential abilities to activate cell-signalling molecules such as Akt and MAPKs, directly involved in cell survival and proliferation [
102]. Functional studies confirm that HPV18 E6 from an African variant has a major effect on the cellular processes including cell cycle and migration [
108]. A specific E6 (amino acid 83) (E6
aa83V) variant is also linked to invasive tumours. The E6
aa83V variant activates PI3K signalling pathway and strengthens the possibility of the existence of Ras-independent mechanisms to recreate signalling through classical Ras effector pathways [
107]. The variant also enhances MAPK signalling and cooperative transformation with deregulated Notch1 signalling. These studies suggest that intra-type genome variations of high risk HPVs may differ in their abilities to mediate Akt /MAPKs signalling, thus presenting a differential threat to the development of cervical and other cancers.
E6 proteins of three HPVs (HPV1, 8 and 16), and BPV1 interact with acidic LxxLL motifs of transcriptional coregulator MAML1 to target many host proteins such as the mammalian target of rapamycin complex 1 (mTORC1) to delay keratinocyte differentiation [
109-
111]. The interaction of HPV-8 E6 with MAML1 causes delay of keratinocyte differentiation [
111]. According to the crystal structure analysis, both BPV1 and HPV16 E6 proteins contain two zinc-finger domains and a linker helix [
109,
110]. Both E6 proteins can bind to LxxLL motifs of the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively to form a basic-hydrophobic pocket. The basic-hydrophobic pocket captures the helical LxxLL motifs to stimulate mTORC1 signalling, and cap-dependent translation, through activation of the PDK1 and mTORC2 kinases leading to genetic alterations [
109,
110]. Such genetic alterations include intra-type genome variations of the virus and changes in chromatin proteins and histone modifications in host cells during HPV16-induced carcinogenesis [
107]. The integrity of LxxLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins [
109,
110].
Generally, β-HPV E6 proteins interact with fewer cellular proteins as is also observed for the α-HPV E6 [
58]. This is because β-HPVs such as HPV5 and HPV8 E6 proteins lack the domains for binding to the LxxLL and PDZ motifs. An exception is that both α- and β-HPV E6 proteins can directly interact with p300 protein, a transcriptional co-activator. The interaction appears to be much stronger with β-HPV 5/8 E6 than with α-HPV 16 E6 or β-HPV 38 E6 [
58]. Enhanced interaction between β-HPV 5/8 E6 and p300 leads to p300 degradation and the blockage of Akt/p300 association in a proteasomal-dependent but E6AP-independent manner [
58]. Decreased p300 concomitantly affects downstream signalling events including expression of differentiation markers K1/10 and involucrin. These results reveal a unique way in which β-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs. Furthermore, HPV16 E6 degrade tuberin, the product of mTOR inhibitor tuberous sclerosis complex 2 (e.g., tumour suppressor gene TSC2), by binding to the DILG motif and ELVG motif located in the carboxyl-terminal of Tuberin, which leads to the phosphorylation of p70 S6 kinase (S6K) [
112-
114]. The E6 binding domain interacting with tuberin is different to that of p53 [
113]. The S6K phosphorylation is tightly associated with HPV16 infection in cervical and oesophageal cancers [
112]. Immunohistochemical analysis of p-S6K
(Thr389) and p-S6
(Ser235/236) in 140 cervical cancer and 161 oesophageal cancer specimens has revealed that both p-S6K and p-S6 were detected significantly more frequently in the HPV16-infected cervical cancer specimens than those in the HPV16-negative specimens [
112]. HPV16 E6 activates S6K via Akt signalling, which promotes S6K phosphorylation and sustains the activity of the mTORC1 and mTORC2 signalling cascade [
112,
115]. Alternatively, HPV16 E6 increases the mTORC1 activity through enhanced phosphorylation of mTOR and activation of the downstream signalling through S6K and eukaryotic initiation factor binding protein 1 (4E-BP1) [
116]. HPV16 E6 also causes Akt activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. HPV16 E6 increases protein synthesis by enhancing translation initiation complex assembly at the 5′ mRNA cap. The increase in cap-dependent translation likely results from HPV16 E6-induced Akt /mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. HPV16 E6-mediated activation of mTORC1 signalling and cap-dependent translation may be a mechanism employed by HPV to promote viral replication in HPV oncoprotein-expressing proliferating cells under conditions of limited nutrient supply [
116].
NHERF-1 is a molecular pathway organizer that plays an important role in a number of cellular processes including signal transduction, cellular transformation and recruitment of membrane, cytoplasmic, and cytoskeletal signalling proteins into functional complexes [
117]. HPV16 E6 mediated-NHERF-1 degradation correlates with the activation of the PI3K/Akt pathway during carcinogenesis [
103]. HPV16 E7 plays a concerted role in E6 mediated NHERF1 degradation [
103]. E7 activates the cyclin-dependent kinase complexes to promote the accumulation of a phosphorylated form of NHERF-1 that is preferentially targeted by E6. However, HPV18 E6 does not degrade NHERF-1, suggesting that HPV E6-induced NHERF-1 degradation is HPV type-dependent [
103]. In addition, E6-upregulated cIAP2 protein confers resistance to cisplatin in HPV 16/18-infected lung cancer through EGFR/PI3K/Akt pathway [
118]. Thus, EGFR or PI3K inhibitor combined with cisplatin may improve the chemotherapeutic efficacy in HPV-induced cancers [
118].
E7 oncogene
HPV E7 protein is responsible for pRb disruption in HPV-induced carcinogenesis. E7 binds to and inactivates pRb to disturb the normal cell division process, allowing the cells to grow out of control and unhindered and thus become cancerous. Clinically, decreased Rb expression is consistently associated with increased CIN grade in the HPV-infected woman’s cervices. It has been reported that HPV E7 significantly up-regulates Akt activity in differentiated keratinocytes, which depends on the ability of E7 binding to and inactivating the proteins of pRb family [
17] . Up-regulation of AKT activity and loss of pRb were observed in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Therefore, pRb expression is inversely correlated with Akt activity in HPV-positive cervical high-grade squamous intraepithelial lesions [
17]. E7 directly activates Akt by phosphorylating it at two key sites (threonine 308 and serine 473), which subsequently leads to phosphorylation of BAD, a downstream target of Akt [
16]. Akt phosphorylation is associated with activated Notch1 signalling that regulates the PI3K pathway [
27,
49]. It has been reported that protein phosphatase 2 (PP2 or PP2A), a ubiquitous and conserved serine/threonine phosphatase, interacts with the 35 kDa catalytic and 65 kDa structural subunits of p-Akt to dephosphorylate Akt [
119]. Akt dephosphorylation results in loss of its activity in preventing cell apoptosis. HPV E7 binds to the two PP2A subunits to prevent their interactions with p-Akt, thereby maintaining Akt signal activation [
16].
Through the PI3K/Akt signalling pathway, HPV E7 oncoprotein inhibits the functions of two cyclin-dependent kinase inhibitors, p21
Cip1 and p27
Kip1 [
120,
121]. As a tumour suppressor, p21
Cip1 binds to the cyclin E/CDK2 complex to maintain Rb in a phosphorylated state [
76]. In the absence of immortalizing oncogenes or genetic lesions, activation of the Raf/Ras pathway results in a p21
Cip1-dependent cell cycle arrest [
122]. In contrast, in the E7-transformed human primary cells, E7 cooperates with Ras to abolish the p21
Cip1-mediated growth arrest [
121]. E7 bypasses Raf-induced arrest and alleviates inhibition of cyclin E-CDK2 without suppressing Raf-specific synthesis of p21
Cip1 or derepressing p21
Cip1-associated CDK2 complexes by sustaining Akt activity [
2,
123,
124]. P27Kip1 is a marker of poor prognosis in several forms of cancer when localized to the cytoplasm and has been implicated as a positive regulator of cellular motility [
120]. HPV 16 E7 protein can modulate the cytoplasmic localization of p27
Kip1 and may in turn regulate tumor metastasis/aggressiveness through the PI3K/ Akt pathway [
120]. E7 also antagonizes the ability of p27
Kip1 to block cyclin E-associated kinase and to inhibit transcription of cyclin A
in vitro [
125].
Apoptosis as a normal process of cellular self-destruction or suicide is one of the major contributors to the development of a normal immune system, which serves a protective role in our bodies. In response to oncogenic insults, normal human cells execute a defence response that culminates in apoptosis [
126]. In HPV infection, expression of E6/E7 oncogens induces cellular immortalization and transformation and carcinogenesis through the immune evasion or resistance against apoptosis and adaptive immune surveillance
. Several studies have reported that activation of Akt induced by HPV E7 expression plays a crucial role in immune resistance [
126-
128]. Due to HPV16 E7 expression, activation of Akt in TC-1/PO and A17 tumours induces an immune resistance against apoptotic cell death [
127]. The E7-induced activation of Akt in A17 tumor cells also contributes to significantly upregulate expression of the key antiapoptotic proteins including Bcl-2, Bcl-xL, phosporylated Bad (p-Bad), Bcl-w, cIAP-2 and surviving [
127]. Treatment of A17 tumor cells with the Akt inhibitor, API-2, reduces the expression of the antiapoptotic proteins markedly leading to an increase in the apoptosis of tumor cells [
128]. It has also been reported that overexpression of E6/E7 from the high-risk HPV16 significantly upregulates expression of cellular inhibitor of apoptosis protein 2 (c-IAP2), which is necessary for the E6/E7-induced resistance to apoptosis and cell survival in HPV16 E6/E7-immortalized human oral keratinocytes [
128]. Akt inhibitors markedly abrogate the antiapoptotic effect of c-IAP2 and some other antiapoptotic proteins on different cancer cells [
129,
130].
Furthermore, normal human diploid fibroblasts expressing the HPV16 E7 oncoprotein are predisposed to apoptosis when they are deprived of growth factors such as IGF-1 in serum-starved medium [
126]. The apoptosis of serum-starved HPV16 E7-expressing cells is directly associated with low phosphorylation of Akt and highly activated caspase 3 that plays a central role in the execution-phase of cell apoptosis. Exogenously added IGF-1 can partially inhibit the cell death response associated with upregulated p-Akt in serum-starved E7-expressing cells [
126]. In support of these previous findings, we observed that HPV16 E7 inhibits IFN-γ-mediated MHC class I antigen presentation and CTL-induced lysis through blocking interferon regulatory factor-1 (IRF-1) expression in mouse keratinocytes [
131]. IRF-1 is a tumor suppressor that can regulate gene expression involved in induction of apoptosis and cell growth control by reducing p-Akt expression [
132]. Thus, the activation of PI3K/Akt pathway induced by HPV E6/E7 oncogenes may represent a new mechanism of immune escape and have important implications for developing a novel strategy in cancer immunotherapy against immune-resistant tumor cells [
127,
128].
As mentioned above, keratinocytes are the host cells of HPV infection. In normal epithelial tissues, cell division and proliferation of keratinocytes are confined to the basal layer, where mitogenic signals are balanced by survival signals transmitted through PI3K/Akt pathway [
133]. Once in the suprabasal layer, keratinocytes stop dividing and enter a differentiation program. Primary keratinocytes in
in vitro cultures resemble
in vivo epidermal development when they enter a differentiation program [
134]. We and others have previously observed that both human and mouse primary keratinocytes grown
in vitro proceed to cell differentiation with downregulation of proliferation markers including K14 and K5 and upregulation of differentiation markers such as involucrin and K10 [
36,
135,
136]. However, expression of HPV 16 E7 in human foreskin keratinocytes in
in vitro cultures induces phosphorylation of AKT on threonine 308 and serine 473 to significantly inhibit cell differentiation and cause hyperproliferation [
16,
17]. It has been reported that a dual epidermal growth factor receptor (EGFR) and HER2 inhibitor Lapatinib reduces expression of E6/E7 and Akt phosphorylation to prevent cell proliferation and induce cell death in HPV-positive cell lines [
137]. The HPV E7-activated Akt also enhances keratinocyte migration through downregulation of RhoA activity [
120]. Either treatment of PI3K or AKT inhibitors or PIK3CA siRNA transfection results in a significant decrease of E7 expression and E7-induced Akt phosphorylation, consequently, leading to that cellular viability and migration are dramatically reduced in HPV16-transfected keratinocytes [
65]. The HPV E7-activated Akt regulates not only tumourigenesis and invasion [
138], but also tumor metastasis/aggressiveness by modulating the cytoplasmic localization of p27 [
120].
E5 oncogene
HPV E5 gene encodes an 83-amino acid, membrane-bound protein, which plays an important role in early cervical carcinogenesis by regulating several cellular pathways [
139-
141]. HPV16 E5 itself cannot immortalize human or mouse primary cells, but can enhance the immortalization of keratinocytes by E6/E7[
142] and potentiate the transforming activity of E7 in murine fibroblasts and activation of EGFR in human keratinocytes that naturally express high levels of EGFR after EGF stimulation [
104,
143-
146].
HPV16 E5 induces the anchorage-independent growth of murine fibroblasts by overexpressing EGFR [
147]. HPV16 E5 also induces expression of VEGF, which plays a central role in switching on angiogenesis during early cervical carcinogenesis through activation of EGFR and phosphorylation of Akt and ERK1/2 [
148,
149]. Thus, HPV 16 E5 may activate the EGFR/PI3K/Akt/MEK/ERK1/2 pathway. Recently, it has been reported that expression of HPV16 E5 in undifferentiated keratinocytes alters the key paracrine mediator of epithelial homeostasis, keratinocyte growth factor receptor (KGFR/FGFR2b) [
146]. KGFR down-modulation, together with a ligand-dependent decrease of p63, is responsible for a E5-mediated decrease of the early differentiation marker K1 and impairment of keratinocyte differentiation [
146].
HPV E5 may act as a survival factor as the E5-expressing cells in human keratinocyte culture exhibit a significant reduction in UVB-irradiation induced apoptosis [
145]. A genome-wide microarray assay reveals that E5 expression significantly alters expression of 179 genes including upregulation of PI3K and PKCδ and downregulation of lamin A/C protein, which lead to inhibition of apoptosis and the establishment of persistent infection in the epithelium [
150]. The E5-mediated protection against apoptosis can be blocked by two specific inhibitors of the PI3K/MAPK pathways (wortmannin and PD98059), suggesting that the PI3K/MAPK pathways are involved in the protection from apoptosis by HPV16 E5 [
145]. Inhibition of the PI3K/Akt signalling prevents the down-regulation of KGFR/p63, supporting an oncogenic role of E5 through the PI3K/Akt pathway [
146]. In addition, two BPV1 E5 mutants are severely defective for focus formation, but still competent for enhanced growth through the PI3K/Akt/cyclin D3 pathway together with a Grb2-Gab1-SHP2 complex and JNK protein [
151,
152]. Thus, it appears that HPV E5 oncoprotein can directly or indirectly target several other substrates to regulate the PI3K/Akt /mTOR pathway.