Introduction

Human papillomaviruses (HPVs) are non-enveloped, epitheliotropic, circular double-stranded DNA viruses [1,2]. HPV infection leads to many different cancers [1,3-5]. It has been well established that high-risk sexually transmitted HPVs such as HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 may lead to the development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and anal intraepithelial neoplasia and squamous cell carcinoma (AIN) while cutaneous β-HPVs such as HPV 2, 4, 5, 8, 10 and 15 are suspected to have an etiologic role in skin warts and cancers [3,6-8]. In total, an estimated 5% of human cancers are caused by HPV infections [9].

Genomic instability is a hallmark of carcinogenesis and recognised as an important factor in the accumulation of mutated genes required for carcinogenesis [10]. Endogenous mutations and accumulation of mutational events are very important in the pathogenesis of premalignant lesions and tumour progression, which promote genomic instability to decrease the ability of maintaining the fidelity of DNA sequences [3,11,12]. Published studies have shown that HPV infection causes genomic instability (chromosomal gain or loss) and gene alterations including endogenous mutations and increased DNA damage which are associated with cancer development [13,14]. In HPV-positive cervical and vulva squamous cell carcinomas, the most common lesions were the loss of 11q and gains of 3q, the latter has been found in more than 25% of high grade CIN [13]. A more comprehensive understanding of genomic instability and mutational events associated with the development of cancers caused by HPV infection is needed and will be separately discussed.

It has been well established that HPV E6 and E7 oncogenes inactivate two tumor suppressors (p53 and pRb) in virus-infected cells. Molecular and cell biology approaches have revealed that alterations of additional signalling pathways are equally important for transformation of HPV oncogene-transduced cells [15]. It is now widely accepted that PI3K/Akt/mTOR signalling pathway plays a pivotal role in many human cancers. HPV infection accompanied by E6/E7 expression activates this signalling pathway by altering multiple cellular and molecular events to drive carcinogenesis [16-18]. The PI3K pathway is unique, in that all of the major components of this pathway have been found to be frequently amplified or mutated in HPV-induced cancers [19-24]. The PI3K/Akt/mTOR signalling pathway mediates the multiple cellular and molecular functions through the altered expression of its targeted genes, which are critical to tumor initiation, progression and outcomes [25,26]. Thus, this pathway has been proposed as a promising therapeutic target for many cancers including cervical cancer [27]. In this review, we summarise the current knowledge of the roles of the PI3K/Akt/mTOR signalling pathway in HPV-induced cancers.

HPV life cycle and ATM /p38MAPK/MK2 pathways

HPVs whether they are low-risk and high-risk are epitheliotropic. Infection with two HPVs may be latent or active [28]. The latent HPV infection will complete the viral life cycle to produce virus progeny, which arises via the distinctly different mechanisms from those involved in active HPV infection [29]. During the HPV life cycle, genome amplification is necessary for production of the virus progeny that is prevented until the levels of viral replication proteins rise, and depends on the co-expression of several viral proteins [30]. Expression of E6 and E7 in the lower epithelial layers drives cells into S-phase creates an environment that is conducive for viral genome replication and cell proliferation [31,32]. The lower epithelial layers where HPVs can establish their infection are the only compartment to contain the cells progressing through the cell cycle [33]. Viral capsid proteins (L1 and L2) are expressed to assemble the virus progeny in cells upon their differentiation that also express E4 to complete its life cycle when the infected cell enters the upper epithelial layers [29]. We have confirmed that expression of HPV6b and BPV1 L1 proteins is dependent on cell differentiation in primary keratinocyte culture systems [34-37]. Thus, the late phase of HPV life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment [38].

It has been established that the cellular DNA damage response (DDR) is activated during the HPV life cycle [39]. This activation leads to the induction of an Ataxia-telangiectasia mutation (ATM)-dependent signalling cascade, DNA repair and cell cycle arrest during G2/M to avoid further DNA damage [15,30,34,38,40-42]. Thus, G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented the virus infection [33]. The ATM pathway is responsible for the DDR to double-strand DNA breaks, which is mediated through the action of downstream kinases, such as CHK2 [39,42,43]. The E1 gene might play a key role in this process, which causes double-strand DNA breaks in the host genome [40,44]. By activating the ATM pathway, HPV recruits cellular DNA repair and recombination factors into its replication centers during the stable and vegetative phases of its life cycle [45]. In cells with impaired p53 activity, DNA damage repair requires the activation of p38MAPK along with MAPKAP kinase 2 (MK2) [43]. In HPV-positive cells, phosphorylation of p38 and MK2 proteins was induced along with relocalization to the cytoplasm. Treatment with MK2 or p38 inhibitors blocked HPV genome amplification, confirming the p38/MK2 pathway as a key regulator of the HPV life cycle [43]. Thus, it appears to be clear that the ATM/p38MAPK/MK2 pathways are required for HPVs to complete normal life cycle in the host body.

HPV infection, carcinogensis and PI3K/Akt/mTOR signalling pathway

Active HPV infection which is also known as abortive infection leads to induction of cancer including benign and malignant neoplasms [46]. In the case of carcinogenesis, viral infection induces the initiation and development of cervical and other cancers via their interactions with different cellular signalling pathways in host cells [47]. In addition to the inhibition of p53 and pRb, HPVs also interact with four major upstream pathways (growth factor receptor, notch receptor, Ras and PI3KCA genes) to stimulate host cell survival and proliferation, leading to carcinogenesis through activation and alteration of the components of the PI3K/Akt/mTOR pathway [19,48-53] (Figure 1).

Figure 1
figure 1

HPVs infect host epithelial cells (keratinocytes) by interacting with different cell surface receptors such as integrin and heparan sulfate proteoglycans (HSPGs). The HPVs replicate themselves using the host cell replication apparatus to express E6/E7/E5 oncoproteins to immortalize the infected cells not only by inhibiting tumour suppressors p53 and Rb and decreasing apoptosis, but also importantly by activating the PI3K/Akt/mTOR signalling pathway. All these processes enhance cell proliferation leading to the carcinogenesis. Solid line: stimulatory influence; Dashed line: inhibitory influence; ⊗: defective process.

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PI3K

PI3K modulates different signals to prevent apoptosis and promote cellular survival and proliferation in a wide variety of cell types [54,55]. It has been shown that PI3K is amplified and activated in HPV-induced cervical cancers and other cancers [56]. For instance, PI3K activity is significantly increased in laryngeal papilloma (a hyperplastic tumor of the respiratory tract induced by HPV 6/11), leading to upregulation of EGFR and subsequently activation of MAPK/ERK [57,58]. The activation of MAPK/ERK in turn alters transcription of multiple genes that are important for cell cycle regulation and cell proliferation. Furthermore, HPV infection causes laryngeal epithelial cells to develop recurrent respiratory papillomas where expression of keratin-13 (K13), a cell differentiation marker of human mucosal keratinocytes, is diminished [58]. This is due to the increased PI3K activity that enhances papillomas cell proliferation and represses terminal differentiation (and hence the failure to upregulate K13 expression) [58].

Liu et al. reported that BPV-1 L1 and L2 pre-mRNAs are spliced in keratinocytes, which contain two exonic splicing enhancers [59]. Each exonic splicing enhancer has an alternative splicing factor (ASF) and splicing factor 2 (SF2), which together play an important role in viral RNA expression and splicing at the proximal 3′ splice site [59]. Depletion of ASF/SF2 from the cells greatly decreases viral RNA expression and RNA splicing at the proximal 3′ splice site. Activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells are mediated through the PI3K/Akt pathway and associated with the enhanced expression of other serine/arginine-rich (SR) proteins [59]. The ASF/SF2 co-operate with H-Ras to enhance cellular proliferation and protect cells from apoptosis by upregulating expression of phosphorylated SR proteins (SRp30s and SRp40) through the PI3K/Akt pathway in cervical and other cancers [59,60]. A published study has also reported that HPV infection-induced IL-17 expression can stimulate Mcl-1 expression to promote lung tumor cell progression through the PI3K pathway [61].

Akt

Akt is a serine/threonine-specific protein kinase, which plays a key role in multiple cellular processes including apoptosis and cell proliferation. Phosphorylation and activation of Akt also play an important role in the majority of HPV-caused malignancies including anal squamous cell carcinomas (ASCCs) [7]. Sixty six percent (82/125) of ASCCs show cellular accumulation of p-Akt associated with nuclear accumulation of MDM2 [7]. Thirty nine out of 46 formalin-fixed cervical neoplastic specimens showed p-Akt on serine 473 [62]. Forty-eight percent (12/25) of cervical cancer patients at stage Ib2-IIb exhibited Akt activation in cancer cells [63]. The radiation sensitivity of HPV-infected H&N cancers also correlates to Akt activation [64]. Mechanistically, HPV infection causes genome instability due to integration of the viral DNA into the host genome. Thus, mutations of PIK3CA gene (which encodes PI3K) in HPV-infected cells and tissues or HPV-DNA transformants may account for Akt activation present in cervical and other types of HPV-induced cancers, since PIK3CA shows the highest frequency of gain-of-function mutations in these cancers [20,63,65]. Oncogenic mutations and amplification of PIK3CA activate PI3K/Akt signalings to drive the HPV-induced tumorigenesis [19,21,65].

Akt phosphorylation is required for a BRCT (BRCA1 carboxyl-terminal) domain-containing protein TopBP1 to interact with other transcription factors, especially E2F1. E2F1 functions as a tumor suppressor to induce apoptosis [66,67]. Phosphorylated Akt (p-Akt) interacts with E2F1 to repress its proapoptotic activity and induce oligomerization of TopBP1 [66]. Furthermore, an endosomal/lysosomal cysteine protease cathepsin L (Ctsl) has been reported to act as an anti-tumor protease. Ctsl is critical for termination of growth factor signalling in the endosomal/lysosomal compartment of keratinocytes and has an inhibitory role in Akt activation in HPV-induced carcinogenesis [68,69]. Therefore, activation of Akt/MAPK pathway is only upregulated in Ctsl-deficient cells [68]. Additionally, papillomavirus-binding factor (PBF), a transcriptional regulator, controls the promoter activity of HPVs by binding to the regulatory sequences of certain papillomavirus types and Huntington’s disease binding protein 2 (HDBP2) through the 14-3-3β protein via two motifs (RSRSLSFSEP and LSKSAP) [70,71]. Activated Akt phosphorylates the two motifs, allowing PBF to associate with 14-3-3β to promote cell survival and growth [70]. These studies suggest that activation of Akt may contribute to the HPV-induced tumorigenesis. It has been reported that high levels of p-Akt might be an unfavourable prognostic marker for relapse-free survival in oropharyngeal cancer [51].

Mammalian genomes contain three Akt genes, Akt1, Akt2 and Akt3. Akt1 encodes the principal Akt isoform that regulates apoptosis [72]. HPVs may differentially affect epithelial Akt activity, as the three Akt isoforms behave differently during epidermal tumorigenesis [73,74] . Cutaneous HPV8 early genes reduce epidermal Akt activity primarily due to down-regulation of Akt1. In contrast, Akt activity can be focally stimulated by up-regulation and phosphorylation of Akt2 in both papillomas and HPV gene-induced epidermal tumours. In SCC, Akt1 is commonly down-regulated consistent with a viral influence, whereas Akt2 up-regulation is widespread. Activation of upregulated Akt2 by serine phosphorylation associates with high-grade tumours, and is characteristic of SCC associated with malignancy [74]. Interestingly, high level of Akt2 is often associated with the presence of β-HPV species (HPV 15) and the up-regulation of p16INK4a and Akt/PI3K pathways [51]. p-Akt2 is over-expressed in basal cell carcinoma (BCC) accompanied by up-regulation of tumor suppressor p16INK4a [51,75]. Overexpression of p16INK4a is common in cervical cancer where pRb protein is inactivated by high-risk HPVs. However, it is still unclear whether p16INK4a overexpression can be used as an indicator of pathogenic activity of high-risk HPVs. Nevertheless, the PI3K/ Akt /mTOR pathway is associated with the up-regulation of p16INK4a by HPVs [19,65,75-79]. So far, it remains unclear whether and how Akt 3 plays a functional role in HPV-induced tumorigenesis.

mTOR

mTOR kinase acts as a cellular rheostat that integrates the signals from a variety of cellular signalling pathways to sense growth factor, nutrient availability and energy status. Recently, it has been reported that activation of Akt /mTOR can be detected within several minutes following exposure of human keratinocytes to HPV16 pseudovirions [80]. mTOR activation is frequently observed in cervical squamous cell carcinoma, most HPV(+) head and neck squamous cell carcinomas (HNSCC), HPV(+) oropharyngeal cancers (OPSCC), cervical cancer squamous cell carcinomas (CCSCC) lesions and cell lines [2,17,63,81]. A tissue microarray analysis has shown that 13 cervical cancer patients (52%) express phosphorylated mTOR (p-mTOR) in the cytoplasm and membrane of cancer cells [63]. Both p-mTOR expression and distant metastasis significantly correlate with the response to nucleus accumbens core [63]. Another analysis of 20 samples each of normal cervix, high-grade squamous intraepithelial lesions (HSIL) and invasive SCCs, derived from a total of 60 cases of cervical biopsies and cervical conizations, has revealed an increased nuclear translocation of both p-mTOR(Ser2448) and p70S6K(Thr389), indicating the constitutive activation and overexpression of the mTOR pathway in HSIL and SCC [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.

HPV pseudovirions and PI3K/Akt /mTOR signals

It is well documented that induction and progression of tumours by HPV infection are driven by the continuing expression of E6 and E7 oncogenes that degrade and inactivate p53 and pRb, respectively [122,153]. However, two studies have reported that HPV pseudovirions and virus-like particles (VLPs, or as pseudovirions), which do not contain E6/E7 genes or their protein products, also can activate PI3K signalling in human keratinocytes and epidermoid carcinoma cells through the signals of growth factor receptor (GFR) [80] and α6β4 integrin receptor [154]. The pseudovirions-induced PI3K activity results in efficient activation of its two down streamers Akt and mTOR and subsequent phosphorylation of the mTOR complex 1 substrates 4E-BP1 and S6K [80] and of FKHR and GSK3β (Figure 2) [154]. These events combined with activation of Ras/MAPK to enhance cell proliferation and inhibit autophagy [80,154].

Figure 2
figure 2

HPV pseudovirions enter the host epithelial cells (keratinocytes) by interacting with α6β4 integrin receptor [154] or growth factor receptor [80] to enhance cell proliferation through the activation of the PI3K/Akt/mTOR and PI3k signalling pathway. Without the oncogenic roles of E6/E7E5, it is impossible for HPV pseudovirions to induce formation of cancerous cells by activating the PI3K/Akt signalling pathway.

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Generally, it is impossible that attachment of HPV pseudovirions and other viruses to the surface of cells activates the PI3K/Akt signalling pathway leading to the development of cancerous cells. However, several other published studies appear to suggest that PI3K signalling pathway plays a critical role in cellular entry of pseudovirions of HPV [155] and other viruses such as Zaire Ebola virus (ZEBOV) [156], SV 40 [157] and Epstein–Barr virus (EBV) [158]. Infection with either radiation-inactivated ZEBOV virus or SV40 VLPs activates PI3K/Akt by expression of phosphorylated PI3K/Akt in the infected cells [157,158]. On the other hand, inhibition of PI3K significantly reduces infection rate of HPV 16 pseudovirions (50–60% reduction) [155] and ZEBOV VLPs [156]. In addition, EBV latent membrane protein 2A can transform the EBV-infected cells to become cancerous through constitutive activation of the PI3K/Akt/Ras/MAPK pathway [158]. Nontheless, whether the PI3K/Akt pathway can play a role in HPV-induced carcinogenesis independent of E6/E7 proteins remains questioned. Thus, more detailed studies are required to improve our current understanding of the full spectrum of mechanisms underlying HPV-induced carcinogenesis.

Concluding remarks

Recently, the PI3K/Akt/mTOR pathway has been identified as an important signalling pathway to tightly modulate many cellular events including the physiological activities of mitogenic or oncogenic factors, leading to the genesis of many human cancers. Published studies have shown that expression of HPV E6/E7 oncoproteins induces HPV transformed cells to be cancerous not only causing degradation and destabilization of p53 and pRb, but also altering multiple cellular and molecular events through activation of the PI3K/Akt/mTOR signalling pathway. The PI3K/Akt/mTOR signalling pathway in HPV-infected cells is activated through both mutation of the pathway components and activation of upstream signalling molecules. Activation of this pathway contributes to genetic instability, deregulation of proliferation, resistance to apoptosis, and changes in metabolism characteristics, eventually leading to the malignant transformation of the infected cells. This signalling pathway may potentially represents both a great therapeutic opportunity and a practical challenge for treating HPV-induced cancers. Thus, further understanding of the molecular mechanisms by which HPV infection activates the PI3K/Akt/mTOR signalling pathway and the biological roles of this pathway in HPV-induced carcinogenesis will improve the disease prevention, patient care, and surveillance strategies for HPV-positive cancers. We suggest that one important research direction will be to devise the novel biomarker-driven therapeutic strategies to target the PI3K/Akt/mTOR pathway in HPV-associated cancers with a specific molecular profile and evaluate the efficacy of the potential therapeutic agents.