Signaling pathways in HPV-associated cancers and therapeutic implications.

Reviews in Medical Virology

REVIEW

Rev. Med. Virol. 2015; 25: 24–53. Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/rmv.1823

Signaling pathways in HPV-associated cancers and therapeutic implications Jiezhong Chen* School of Biomedical Sciences and Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia

S U M M A RY Human papillomaviruses (HPVs) are small double-stranded circular DNA viruses with 8 kb genomes. So far, more than 150 HPVs have been identified, and 12 types of HPVs have been conclusively linked to cancer by the International Agency for Research on Cancer/World Health Organization. Expression of HPV E5, E6 and E7 oncoproteins can alter multiple signaling pathways to cause cancer. In this review, the signaling pathways activated by these oncoproteins are summarized, and targeted therapy against key signaling molecules is described. E6 can inactivate tumor protein 53 and PDZ (post synaptic density protein–drosophila disk large tumor suppressor–zonula occludens-1 proteins) while stimulating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), Wnt and Notch pathways. E7 can inhibit retinoblastoma protein and stimulate the PI3K/Akt pathway. Both E6 and E7 can deregulate cellular microRNA expression, which can alter cellular signaling pathways. E5 can sensitize epidermal growth factor receptor to epidermal growth factor to increase activation of PI3K/Akt and mitogen-activated protein kinase pathways. E5 can also inhibit the extrinsic apoptotic pathway. These altered signaling pathways could be critical for the initiation and maintenance of HPVassociated cancers. Therefore, targeted therapy against the key signaling molecules has therapeutic implications. Among these, the possibilities of targeting PI3K/Akt, mammalian target of rapamycin, epidermal growth factor receptor and vascular endothelial growth factor have been extensively studied in many cancers. Some inhibitors have been studied in cervical cancer in both animal models and clinical trials. Although the results are promising, further investigation is warranted. Copyright © 2015 John Wiley & Sons, Ltd. Received: 17 May 2014; Revised: 15 October 2014; Accepted: 27 December 2014

*Correspondence to: J. Chen, School of Biomedical Sciences and Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. E-mail: [email protected] Abbreviations used Akt, protein kinase B; BMI, B cell-specific Moloney murine leukemia virus integration site 1; CBF-1, C-promoter binding factor 1; CDK2, cyclin-dependent kinase 2; COX-2, cyclooxygenase-2; CSCs, cancer stem cells; CSL, CBF-1-Su (H) and LAG-1; CTGF, connective tissue growth factor; CYR61, cysteine rich 61; DKK-1, dickkopf-related protein family; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; E6AP, E6-associated protein; FZD7, frizzled family receptor 7; GIPC, GAIP-interacting protein, C terminus; GSK3b, glycogen synthase kinase 3b; Hes, hairy and enhancer of split; Hey, Hes-related repressor protein; HPV, human papillomavirus; Lag-1, lobster agglutinin 1; LIM, Lin11, Isl-1 and Mec-3; MAGI-1, Membrane-associated guanylate kinase, WW and PDZ domaincontaining protein 1; miRNAs, mircroRNAs; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor-κB; NFX1, nuclear transcription factor, X-box binding 1; NICD, Notch intracellular domain; p53, tumour protein 53; PDK1, putative 3-phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; pRb, retinoblastoma protein; PTEN, phosphatase and tensin homologue; STAT-1, signal transducer and activator of transcription 1; TIP-2, Tax-interacting protein-2; VEGF, vascular endothelial growth factor.

Copyright © 2015 John Wiley & Sons, Ltd.

INTRODUCTION Human papillomaviruses (HPVs) are a family of small double-circular-stranded DNA viruses with genomes containing 8 kb DNA sequences [1]. The sequences of some HPVs, such as HPV16 and HPV18, which are the most common cancer-related HPVs, encode two late genes L1 and L2 and six early genes E1, E2, E4, E5, E6 and E7. Although E1, E2 and L1 and L2 proteins are essential for all HPVs, expression of other HPV proteins varies in different HPVs. For example, HPVs 101, 103 and 108 do not encode E6 [2], while HPV31 expresses E8 [3]. These HPV proteins maintain replication, amplification and release of the viruses. Among them, E5, E6 and E7 are major oncogenes that promote host cell proliferation to facilitate viral amplification. E6 and E7 can be integrated into host genomes in some cases and expressed in high levels, while E5 can only be expressed from viral genomes and maintained as episomes in host cells [4,5]. Infections with some HPVs cause cancers

Signaling pathways in HPV-associated cancers including cervical cancer, head and neck cancer, vulvar cancer, vaginal cancer, penile cancer and anal cancer. So far, more than 150 types of HPVs have been identified. Among them, 12 types of HPVs can cause cancer (http://monographs.iarc. fr/ENG/Monographs/vol90/index.php), and the list could be expanded with more cancer-related HPVs identified. HPV16 and HPV18 are responsible for 50% and 20% of cervical cancer respectively [5]. Although HPV infections are very common, only a very small percentage of HPV-infected people develop cancer. HPV infections elicit immune responses, which clear most HPV viruses. Persistent infections greatly increase the risk for carcinogenesis. Understanding the mechanisms of HPVcaused cancer could be helpful for the prevention and treatment of the disease. Differences in the sequences caused by amino acids as well as variation in codon usage of E5, E6 and E7 genes have been associated with their ability to initiate cancer [6–9]. A study has shown that E7 is sufficient to immortalize cells, while E6 can increase the effect of E7, that is E6 and E7 can cooperate to immortalize primary human epithelial cells. The complementary effects of E6 and E7 could be explained by their different downstream targets. Inhibition of E6 led to increased tumor protein 53 (p53) but not retinoblastoma protein (pRb), while inhibition of E7 resulted in increased pRb but not p53 [10]. E5 is also an oncoprotein, which promotes carcinogenic signaling pathways to increase the effects of E6 and E7. However, E6 and E7 together are not sufficient, requiring introduction of another cancer risk factor for tumor formation. For example, oncogene Ras has been shown to cause cancer together with E6 and E7 [11]. Deletion of tumor suppressor gene RXRα and expression of E6 and E7 together have also been shown to cause malignant lesions of cervical tissue [12]. Many studies have been performed to elucidate the mechanisms for E6 and E7 to cause cancer, showing their interactions with many signaling molecules. In this review, the effects of these oncoproteins on intracellular signaling pathways are summarized in association with the roles of these signaling pathways in carcinogenesis and cancer progression. Several microRNAs (miRNAs) altered by E6 and E7 and associated cell biological activities are also described. After a discussion of the changed pathways on cell behaviors including Copyright © 2015 John Wiley & Sons, Ltd.

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Figure 1. Signaling molecules altered by E6 protein. Human papillomavirus E6 inhibit tumor protein (p)53, PDZ proteins and some microRNAs (miRNAs). E6 activates protein kinase B (Akt), Notch and Wnt pathways. E6 also increases telomerase activity. PDZ, post synaptic density protein–drosophila disk large tumor suppressor– zonula occludens-1 protein

proliferation, apoptosis, genomic instability, migration and drug resistance, the outcomes of targeted therapy against several altered signaling molecules are described. HUMAN PAPILLOMAVIRUS E6 PROTEIN Human papillomavirus E6 is a small protein without enzymatic activity. For example, HPV16 E6 consists of 151 amino acids. E6 protein activates several carcinogenic pathways and inhibits tumor suppressor protein p53 and post synaptic density protein–drosophila disk large tumor suppressor–zonula occludens-1 protein (PDZ) (Figure 1). The survival pathways activated by E6 include phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), Wnt and Notch. E6 activates telomerase expression and activity, thereby inhibiting telomere shortening and increasing cell immortalization. Several miRNAs have been altered by E6, leading to signaling pathway changes.

Inactivation of p53 protein Protein p53, encoded by TP53 gene, is a wellknown tumor suppressor, which prevents cancer initiation by maintaining genomic stability [13,14]. It is activated by DNA damage and environmental stimuli such as oxidative stress and osmotic shock as well as viral infections [13,14]. Activated p53 can slow down the cell cycle to allow damaged DNA to be repaired. Activated p53 binds to DNA to increase expression of protein p21, which inhibits cyclin-dependent kinase 2 Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

26 (CDK2) to decrease G1/S transition. Activated p53 can also initiate apoptosis if DNA is severely damaged. Through these processes, p53 prevents accumulation of cells with DNA damage so that it decreases tumor initiation. In contrast, inactivation of p53 results in the accumulation of abnormal cells that have gene mutations, facilitating cancer initiation. E6 protein inactivates p53 and thus avoids apoptosis of host cells that have DNA damage. Highrisk type HPV E6 binds to an LXXLL motif (where L = leucine and X= any amino acid) on cellular E3 ubiquitin ligase E6-associated protein (E6AP) [15– 17]. The formed E6-E6AP complex then recruits and ubiquitinates p53, which facilitates proteasome-mediated degradation of p53 (Figure 2) [18,19]. This E6-induced p53 decrease has been recognized as a major mechanism for E6 to cause cancer. Inactivation of p53 introduced by dominant negative p53 is sufficient to maintain HPV DNA with E6 inactivating mutations in human keratinocytes [20]. In organotypic cultures, an E6 null mutant accumulated high levels of p53, and

J. Chen HPV amplified very poorly [21]. Silencing of p53 or expression of ectopic wild type E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. E6 can degrade p53 in E6AP-null mice indicating that other ubiquitin ligases may also be involved in E6mediated p53 degradation [22]. The ability of E6 to degrade p53 has been correlated with HPV’s ability to cause cancer. Mesplede et al. examined the p53 degradation ability of E6 proteins from 29 types of HPVs [23]. It was found that variation of the p53 degradation ability was more than 100 times with high-risk HPVs having a higher ability to degrade p53. It was also found that the variants of a type of HPV such as HPV16 and HPV33 had different p53 degradation ability but the difference was much less than in different types of HPVs. While it is certain that degraded p53 by HPV E6 via E6AP plays a critical role in HPV-associated cancer, other alternatives have also been revealed. Studies reported that HPV E6 also degraded other two members of p53 family p63β and p73 [24,25]. These two proteins have similar structures to p53 and also can inhibit cell proliferation and promote apoptosis [26]. Thus, inactivation of p63β and p73 is important for cancer development [27]. Park et al. has shown that E6 directly binds to and inactivates p73, but it is not via E3 ubiqitination [25].

Inhibition of post synaptic density protein– drosophila disk large tumor suppressor– zonula occludens-1 proteins

Figure 2. E6/p53 pathway. Tumor protein (p)53 family members can maintain genomic stability through decreasing cell cycle and increasing apoptosis under stimulation such as ultraviolet (UV), hypoxia and viral infections. Human papillomavirus E6 can bind to ubiquitin ligase E6-associated protein (E6AP) or other ligases to degrade p53. E6 can also act on p63 and p73 directly to destabilize these proteins


The PDZ is a structural domain of 80–90 amino acids, which is shared by many proteins and named from the initial letters of three proteins— post synaptic density protein (PSD95), drosophila disk large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) [28]. There are now more than 250 proteins that have a PDZ domain. PDZ proteins interact with c-tail amino acids of targeted proteins to regulate multiple biological processes, including cell adhesion, tight junction, cell polarity, cytoskeleton, ion channels and signaling pathways. Some PDZ proteins are tumor suppressor proteins, and thus, loss of these proteins facilitates cancer formation [29]. High-risk types of HPV E6 proteins have a PDZ-binding motif at their extreme carboxy termini [30–32]. Binding of E6 to PDZ proteins results in dysfunction of these proteins. Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

Signaling pathways in HPV-associated cancers Both HPV16 and HPV18 E6 bind to Dlg Cterminus for transformation and mutation of E6 loses the ability to bind and transform rodent cells [30,31]. HPV18 E6 protein interacts with TIP-2/ GIPC, leading to polyubiquitination and proteasome-mediated degradation of the protein [33]. In HeLa cells, RNAi silencing of E6 decreases TIP-2/GIPC levels. TIP-2/GIPC can increase expression of the TGF-beta type-III receptor at the cell membrane and thus increase the antiproliferative effect of TGF-beta. Depletion of TIP-2/GIPC in HeLa cells reduced the antiproliferative effect of TGF-beta, while silencing of E6 blocked the proliferation of HeLa cells. HPV E6 acts on PDZ-partitioning defective 3 protein, which regulates tight junctions and cell polarity [34,35]. E6 caused translocation of PDZpartitioning defective 3, leading to the loss of cell polarity to facilitate tumor formation. Massimi et al. showed that E6 targeted hDlg, hScrib (Scribble), MUPP1, membrane-associated guanylate kinase, WW and PDZ domaincontaining protein 1 (MAGI-1), MAGI-2 and MAGI-3 through E6AP to increase transformation [35,36]. HDlg and MAGI-2 interact with phosphatase and tensin homologue (PTEN), which regulates the PI3K/Akt pathway [37,38]. E6 has high affinity for the PDZ domain of MAGI-1 protein, and mutation of the residue lysine 499 abolished the effect of E6 on MAGI-1 [39].

Activation of phosphoinositide 3-kinase/protein kinase B The PI3K/Akt pathway is a major cancer survival pathway (Figure 3) [40,41]. PI3K regulates Akt and Rac-1. Akt has a broad range of downstream targets, which control cell proliferation, cell growth, cell mobilization, angiogenesis and cell survival [40,41]. The pathway has been associated with increased cancer initiation, progression, metastasis and drug resistance. Thus, inhibition of the pathway has been proposed for the treatment of cancer [42]. Targeted therapy of PI3K/Akt has been studied extensively in many cancers such as breast cancer, melanoma, colon cancer and prostate cancer [40,43,44]. Both genetic defects and environmental factors are involved in activation of the pathway. Gene defects have been detected in almost all elements of this pathway [42]. Obesity has been shown to increase this pathway, leading to increased cancer incidence [45,46]. Copyright © 2015 John Wiley & Sons, Ltd.

27 Several studies have shown that E6 can activate this pathway through various mechanisms. E6 inactivates PTEN through PDZ proteins, leading to increased pAkt as well as increased cell proliferation [37,38,47]. The mammalian target of rapamycin (mTOR) kinase is a downstream target of Akt and also activated by blood levels of amino acids and mitogen-activated protein kinase (MAPK) pathway. It has been demonstrated that mTOR is activated by E6 as indicated by increased ribosomal protein S6 kinase, which is regulated by mTOR [48,49]. E6-E6AP complex binds and degrades the mTOR inhibitor tuberous sclerosis complex 2 (TSC2). Another study also showed that HPV16 E6 expression caused an increase in mTOR complex 1 activity indicated by enhanced phosphorylation of mTOR as well as its downstream targets ribosomal protein S6 kinase and eukaryotic initiation factor binding protein 1 under conditions of nutrient deprivation [50]. However, a decrease in TSC2 levels in HPV16 E6-expressing cells was not detected. Instead, upstream kinases putative 3-phosphoinositide-dependent kinase 1 (PDK1) and mTOR complex 2 were activated, leading to increased phosphorylation of Akt [50]. In human foreskin keratinocyte (HFK) cells, expression of HPV16 E6 resulted in sustained activation of receptor protein tyrosine kinases including epidermal growth factor receptor (EGFR), insulin receptor beta and insulin-like growth factor receptorbeta, which are upstream of the PI3K/Akt pathway [51]. E6 can also increase signaling adaptor protein growth factor receptor-bound protein 2 to activate the PI3K pathway [51]. Activation of Akt can produce a cascade of changes in downstream targets. Akt can phosphorylate E6 to promote its ability to interact with protein 14-3-3σ, which is important in carcinogenesis [52]. HPV has also been associated with increased expression of c-myc, a downstream protein of Akt [53–56]. E6 has been reported to act directly on cmyc, leading to activation of telomerase activity [10,57]. Controversially, two studies showed that E6 accelerated c-myc degradation [58,59]. While it is certain that E6 can cause activation of the PI3K/Akt pathway and its downstream targets nuclear factor-κB (NF-κB), mTOR, 14-3-3 and c-myc, the effects of E6 on other downstream targets of Akt are largely not studied. It will also be interesting to investigate the biological effects mediated by the E6/PI3K/Akt pathway Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

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Figure 3. E6/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. PI3K catalyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3), which promotes (PDK1) to phosphorylate Akt. Activated Akt can influence cell survival through mitochondrial Bcl-2 family members, cell proliferation through cyclin D1 and nuclear factor-κB (NF-κB), cell growth through mammalian target of rapamycin and angiogenesis through vascular endothelial growth factor (VEGF). Akt blocks apoptosis through decreasing tumor protein (p) 53, p21 and p27. Akt also increases genomic instability. E6 can activate PI3K through receptor protein tyrosine kinase or direct interaction with PI3K. E6 can also block tuberous sclerosis complex 1/2 (TSC1/2) to increase mammalian target of rapamycin complex 1 (mTORC1) activity to increase cell growth and can block pro-apoptotic proteins Bad and Bax to decrease apoptosis. PTEN, phosphatase and tensin homologue; RPTK, receptor protein tyrosine kinase; HIF, hypoxia-induced factor; S6K, ribosomal protein S6 kinase; GSK, glycogen synthase kinase; Rheb, Ras homologue enriched in brain; 4E-BP, eukaryotic initiation factor 4E binding protein; MDM2, murine double minute; Foxo1, forkhead box O1; MAPK, mitogen-activated protein kinase; mTORC2, mammalian target of rapamycin complex 2

in terms of cell proliferation, survival, migration and drug resistance.

Activation of the Wnt pathway Wnt ligands and the associated pathway regulate cellular proliferation and differentiation processes and thus play critical roles in normal tissue homeostasis [60] and in pathologic conditions such as cancers [61–63]. Activation of the Wnt pathway results in accumulation of β-catenin, which in turn increases transcription of a broad range of genes to promote cell proliferation. In inactivation status of the Wnt pathway, β-catenin forms a “degradation complex” with other proteins including glycogen synthase kinase-3β (GSK3β), casein kinases, adenomatous polyposis coli and Axin2 and Copyright © 2015 John Wiley & Sons, Ltd.

phosphorylated at serine and threonine residues. Phosphorylation of β-catenin induces its ubiquitination by β-TcRP ubiquitin ligase, leading to degradation. In activation status of canonical Wnt signaling pathway, intracellular Dishevelled protein is phosphorylated and interacts with Axin2, leading to dysfunction of the degradation complex and accumulation of β-catenin. The accumulated β-catenin is translocated into the nucleus and binds members of the T-cell factor/lymphoid enhancer factor family of transcription factors to regulate target genes including c-jun, c-myc [64], cyclin D1 [65], multidrug resistance 1[66], matrilysine [67], Axin2 [68], survivin, vascular endothelial growth factor (VEGF), COX-2 and matrix metalloproteinases [69]. It also targets positive and Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

Signaling pathways in HPV-associated cancers negative regulators of the Wnt pathway including Axin2, Wg, FZD7, DKK-1 and sFRP-2 to form autoregulation [69]. Accumulated nuclear β-catenin has been commonly found in human HPV16-positive invasive cancer samples but also in early dysplastic lesions, indicating activation of the Wnt pathway by HPV oncogenes [70,71]. The nuclear accumulation of βcatenin correlates with tumor progression in cervical cancer patients [72]. The accumulated β-catenin also correlates with HPV infection in cell lines including HPV16-positive oropharyngeal cancer cell lines 147 T and 090, HPV-negative cell line 040 T and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) [73]. E6 gene silencing in HPV-positive cells reduced nuclear β-catenin substantially, suggesting a critical role of E6 in the activation of the Wnt pathway. The mechanism has been associated with a protein called seven in absentia homologue, which increases proteasomal degradation of β-catenin. E6 can decrease seven in absentia homologue mRNA and protein levels substantially [73]. Lichtig et al. also showed that HPV16 E6 activated the Wnt/β-catenin pathway; the mechanism is independent of the ability of E6 to target p53 for degradation or bind to the PDZ-containing E6 targets but requires E6AP [74]. In vivo mouse experiments showed that E6 expression led to accumulation of β-catenin [75]. Fulllength E6 oncoprotein expression in K14E6 mice enhanced the nuclear accumulation of β-catenin and the accumulation of cellular β-catenin-responsive genes. These effects were not observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice), indicating that the effect of E6 was mediated by the PDZ domain [75]. Although the Wnt pathway may be a possible mediator for increased β-catenin, PI3K/Akt is also well known to cause accumulation of β-catenin through inactivation of GSK3β [76]. Therefore, further studies are needed for a unified explanation of E6-induced β-catenin accumulation.

Activation of the Notch pathway Notch signaling plays important roles in both normal physiological activities such as cell growth, differentiation, immune responses and organ development and various diseases including cancer [77–81]. Increased expression of the Notch pathway has been associated with progression of several malignancies such as prostate cancer, breast cancer, Copyright © 2015 John Wiley & Sons, Ltd.

29 glioma and head and neck cancers. The pathway comprises four Notch receptors (Notch 1–4 in humans), a group of transmembrane proteins, and their ligands (Jagged1, 2 and delta-like ligand 1, 3 and 4) [82,83]. Binding to their ligands on the surface of neighboring cells leads to the cleavage of Notch receptor by γ-secretase and subsequent release of the Notch intracellular domain (NICD) (Figure 4). As the constitutively active domain of the Notch receptor, NICD translocates to the nucleus where it binds to and forms a complex with the transcriptional regulator termed CBF-1-Su (H) and LAG-1 (CSL) and mastermind-like protein, leading to the displacement of co-repressors previously bound to CSL and recruitment of co-activators. The co-activators then induce expression of the target genes, such as the hairy and enhancer of split (Hes) and Hes-related repressor protein (Hey) families. Accumulating evidence indicates that dysregulated NCID can also activate the PI3K/Akt pathway, which in turn causes a cascade of target proteins [84–88]. The expression of Notch 1 increases with progression of cervical cancer; Notch 1 has been detected in invasive cervical cancer but not low-grade cancer [89]. Daniel et al. showed that Notch-1 receptor expression was increased during the progression from cervical intraepithelial lesions to invasive cervical carcinoma [90]. Moreover, main cellular localization of Notch-1 protein changed from cytoplasmic to nuclear with the transition from intraepithelial lesions III to microinvasive carcinoma [90]. The regulation of the Notch signaling pathway by HPV E6 has been demonstrated in cervical cancer cell lines. At late passage but not early W12 cell line, which is an HPV type-16-positive human cervical low-grade lesion-derived cell line, jagged1 is upregulated, while manic fringe is decreased [91]. It was confirmed by an increase of Notch-driven report activity and a decrease of manic fringe promoter-driven report activity in late passage W12. Inhibition of the Notch pathway by expression of manic fringe, dominant-negative Jagged1 or RNAi silencing of Jagged1 inhibits the tumorigenicity of CaSki, an invasive cervical carcinomas-derived cell line. HPV increases the Notch pathway through distinct mechanisms [92]. HPV16 E6 can interact with cellular protein NFX1-123 and increase NFX1-123 protein expression [92]. Overexpression of NFX1-123 in HPV16 E6-expressing keratinocytes increased Notch-1 mRNA levels. Weijzen et al. demonstrated Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

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Figure 4. E6/Notch pathway. Notch receptor is activated by its ligand Jagged1 from another cell and cleaved by gamma secretase to produce Notch intracellular domain (NICD), which translocates into the nucleus to form a complex with CBF-1-Su (H) and LAG-1 (CSL) and mastermind-like protein (maml) to promote transcription of hairy and enhancer of split (Hes) and Hes-related repressor protein (Hey). NECD, Notch extracellular domain; URR, upstream regulatory region

that presenilin-1 mediated the effect of E6 on the Notch pathway in mouse and human primary cell lines transfected HPV16 E6 ([93]). A controversial opinion about the Notch pathway in HPV-associated cancer has been proposed. It was shown that Notch 1 was decreased in HPV-positive cervical cancer cell lines HeLa, C40I, C4-II, SiHa and Caski compared with HPV-negative C33a and primary keratinocytes [94]. However, expression of Notch 2 in all these cell lines was similar to primary keratinocytes. Nevertheless, overexpression of Notch 1 in HPV-positive cells decreased their growth. This indicates that Notch could be a tumor suppressor in cervical cancer as in some cancers such as chronic myelomonocytic leukemia, cutaneous and lung cancers [95–97]. It has been demonstrated that Notch 1 is controlled by p53. Knockdown of p53 by short interfering RNA (siRNA) decreased Notch-1 levels, while activation of p53 by nutlin increased Notch-1 levels in human keratinocyte cancer cell lines [98]. Suppression of the Notch signaling pathway together with activated Ras resulted in cancer cell formation in primary keratinocytes, providing evidence for carcinogenic effect of loss of Notch 1[98]. There were also reports that Notch was lost because of Notch 1 inactivating mutation in 10–15% of patients and the loss could increase head and neck squamous cell carcinomas (HNSCC) carcinogenesis [99,100]. Copyright © 2015 John Wiley & Sons, Ltd.

However, a recent study showed that the Notch pathway was activated in 32% of HNSCC patient samples and only 9% (4 out of 37) had inactivation of the pathway, casting doubt whether inactivation of Notch is required for these cancers [101].

Telomerase activation Telomerase is a ribonucleoprotein enzyme for maintaining telomeric structures at the end of chromosomes, and its increased activity is important for carcinogenesis [102–105]. In normal cells, telomerase activity is very low so that telomeres shorten with every round of DNA replication and become shorter over time, which leads to replicative senescence. Expression of the telomerase catalytic subunit in normal cells increased telomere length, resulting in indefinite cell proliferation [106]. In cancer cells, inhibition of telomerase activity decreased cell proliferation [107]. Telomerase activation is critical for the immortalization of primary human keratinocytes by the high-risk HPV E6 [108]. E6 is able to increase telomerase activity by upregulation of telomerase reverse transcriptase (TERT), which is encoded by the human telomerase reverse transcriptase (hTERT) gene [108,109]. E6 induces the hTERT promoter via interactions with the cellular ubiquitin ligase, E6AP. E6 increases hTERT via NFX1-123. NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

Signaling pathways in HPV-associated cancers telomerase expression [92]. E6 protein also interacts directly with the hTERT protein [110]. Unlike its effect on p53, E6 binding to hTERT protein does not cause protein degradation both in vitro or in vivo but increases telomerase activation by a posttranscriptional mechanism [110].

MicroRNAs involved in E6-mediated signaling pathways MicroRNAs are small non-coding RNAs containing 19–24 nucleotides, which regulate gene expression by targeting transcripts or translation [111]. In humans, a large number of genes are regulated by miRNAs, so miRNAs regulate many biological processes. Studies have shown that many miRNAs are involved in E6-mediated signaling pathways. Martinez et al. used Ambion (Ambion Technologies, Austin, TX, USA) arrays to show that three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA [112]. Another study showed that miRNAs miR-363, miR-33 and miR-497 were upregulated, whereas miR-155, miR-181a, miR-181b, miR-29a, miR-218, miR-222, miR-221 and miR-142-5p were downregulated in HPV-positive head and neck cancer cells [113]. E6 decreases miR-34a [114,115], which is an important cell cycle regulator [116,117]. MicroRNA34a is a target of p53, and the effect of E6 on miRNA-34a is mediated by decreased p53 [114,115]. This is consistent with the fact that miR-34a is reduced in both normal cervical tissue and cervical lesions with high-risk HPV infection [118]. Targeting of HPV16 E6 has been shown to increase miR-34a [119]. One of the targets of miR34a is p18Ink4c [120]. Silencing of E6 or overexpression of miRNA-34a in HeLa cells decreased p18Ink4c, while inhibition of miRNA-34a increased p18Ink4c. Protein p18Ink4c is an inhibitor of CDK4/6, but its role in cervical cancer is not well studied. Other targets of miR-34a have also been revealed. MicroRNA-34a is negatively correlated with CDK4, Sirt1 and myeloblastosis protein [121]. In cell biology, miRNA-34a has been associated with hypoxia increased epithelialmesenchymal transition and docetaxel resistance in breast cancer [122,123]. Martinez et al. showed that miR-218 was particularly reduced in HPV-positive cervical cancer indicated by microarray, real-time PCR and northern blot analyses [112]. Expression of HPV16 E6 but Copyright © 2015 John Wiley & Sons, Ltd.

31 not HPV6 E6 reduced miR-218 expression, while silencing of E6 increased miR-218 expression in cervical cancer cells. LAMB3, a laminin protein, is increased in cervical cancer specimens and identified as a target of miR-218 [113]. Therefore, LAMB3 was increased by E6 [112]. LAMB3 regulates cell differentiation, migration, adhesion, proliferation and survival, so silencing of LAMB3 in cervical cancer cells reduced cell survival and migration [113]. Consistently, overexpression of miR-218 also reduced cancer cell migration and invasion in both HPVpositive and HPV-negative cervical cancer cell lines. E6 decreases miR-125b [124], which inhibits cervical cancer growth and promotes apoptosis via inhibition of the PI3K/Akt pathway [125]. Another study reported that miR-125b was highly reduced in HPV-positive cells [126]. However, a recent study showed that miR-125 was increased in cervical cancer and miR-125b could target the Bak promoter to reduce its expression and thus decrease apoptosis [127]. This discrepancy has been explained by multiple targets of miR-125b with its roles depending on different expression levels of target genes [128]. MicroR-375 is decreased in HPV-associated cancer, which negatively regulates HPV16 and 18 transcripts [129]. MicroR-375 directly acts on E6AP and decreases its activity [129]. Thus, transfection of miR-375 resulted in increased p53, p21 and 14-3-3. In gastric cancer, miR-375 was shown to inhibit PDK1 and 14-3-3zeta directly, and overexpression of miR-375 increased apoptosis via the caspase pathway [130]. E6 could regulate more miRNAs, so the list might increase after more studies are performed. A recent study showed that in organotypic raft cultures of foreskin and vaginal keratinocytes HPV16 and HPV18 changed 13 miRNAs. MicroR-25, miR92a and miR-378 were increased, while miR-22, miR-27a, miR-29a and miR-100 were decreased. The increased miR-25, miR-92a and miR-378 were confirmed in cervical patients’ specimens [131]. The significance of these changes in cellular signaling pathways is not elucidated yet. HUMAN PAPILLOMAVIRUS E7 PROTEIN E7 is a potent cell cycle regulator with 98 amino acids containing three domains of E7 called conserved regions 1–3. It interacts with more than 50 cellular factors [132,133]. The significance of most of these interactions is unknown. However, several Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

32 downstream targets have been identified including its interactions with pRb, the PI3K/Akt pathway and miRNAs.

J. Chen [147]. However, knockout of three Rb family members in mice is not sufficient to cause cervical carcinoma although development of high-grade cervical intraepithelial neoplasia can develop [148].

Inhibition of retinoblastoma protein Dysfunction and inactivation of pRb through various factors have been associated with initiation of many cancers [134–136]. E7 is one factor that inactivates pRb [137,138] (Figure 5). Several mechanisms for E7 to alter pRb signaling have been elucidated [133,139–141]. Retinoblastoma protein is a pocket protein, which binds to transcription factors E2F1-3 to prevent E2F1-3 functions. When E7 binds its pocket and inactivates its function to bind E2F1-3, E2F1-3 is released. E2F1-3 can bind cell cycle regulators DP-1 or DP-2, leading to increased cell cycle from G1 to S, decreased apoptosis and increased genomic instability. HPV16 E7 proteins can destabilize pRb through proteasomal degradation mediated by cullin 2 ubiquitin ligase complex reprogrammed by HPV E7 [142]. A difference in an amino acid in E7 sequence could affect its binding ability and degradation ability, accounting for carcinogenic ability of high-risk or low-risk HPVs [143,144]. E7 also acts on E2F proteins directly. It binds to E2F1 and increases its activity [145]. E7 can also block the transcriptional suppressor activity of E2F6 [146]. E2F6 is usually upregulated by E2F1 to provide a feedback regulation [146]. In addition, E7 also inactivates other pocket proteins including p130 and p107 [137]. Proteins p130 and p107 regulate E2F4 and E2F5, which also play key roles in cell cycle regulation. The effects of E7 on three pRb family members may be synergistic

Figure 5. E7/retinoblastoma protein (pRb) pathway. pRb forms a complex with E2F1 to keep E2F1 in inactivation status. E7 can bind with pRb, allowing the release of E2F1. E2F1 binds to cell cycle regulator DP1, causing increasing cell cycle from G1 to S, decreased apoptosis and increased genomic instability


Activation of phosphoinositide 3-kinase/protein kinase B Several studies have shown that E7 can activate the PI3K/Akt pathway. Menges et al. expressed HPV16 E7 in HFK cells and showed upregulation of Akt activity in organotypic raft cultures [149]. The mechanism of increased Akt activity has been regarded as inhibition of pRb by E7 as follows. First, the ability of E7 to increase Akt activity is correlated with its ability to bind to and inactivate Rb. Second, silencing of Rb by short hairpin RNAs (shRNAs) in differentiated keratinocytes increased Akt activity. Third, increased Akt activity and loss of Rb were also correlated in HPV-positive cervical high-grade squamous intraepithelial lesions. Protein kinase B upregulation by E7 may be mediated by protein phosphatase 2A (PP2A) [150,151]. PP2A is known to inhibit Akt phosphorylation. Pim et al. demonstrated that E7 could bind both the Mr 35 000 catalytic subunit and the Mr 65 000 structural subunit of PP2A to inhibit PP2A activity [150]. Liu et al. found that PP2A mRNA and protein levels were detected in 73% and 53% cervical cancer samples respectively but not in normal cervical tissues [151]. Knockdown of E7 downregulated both mRNA and protein expression of PP2A. E7 negatively regulates p27 via Akt. HPV16 E7 enhanced both the cytoplasmic retention of p27 in HFKs, which was reduced by PI3K/Akt inhibition [152]. A standard wound assay showed that E7 increased the migration of HFKs, which was Akt/p27 dependent. Rho family guanosine triphosphatases (GTPases) play critical roles in cytoskeleton and cell migration. Under extracellular environment stimulation, GTPases can transfer inactive guanosine diphosphate-bound states to the active guanosine triphosphate-bound form, which in turn changes cell morphology and promotes cell migration through Rho family members [153–156]. HPV E7 regulates Rho family members to increase cell migration through Rac1 and RhoA [152,157]. RhoA is crucial for efficient cell migration and cell spreading. Todorovic et al. used mass spectrometry identifying p190RhoGTPase-activating protein (p190) as a binding partner of HPV16 E7 [157]. Protein Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

Signaling pathways in HPV-associated cancers p190, one of the GTPase-activating protein families stimulated the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and increased cell migration.

MicroRNAs altered by E7 E7 decreases miR-203 during keratinocyte differentiation, which is a tumor suppressor and thus increases carcinogenesis [158]. Recently, several other targets of miR-203 have been identified. MicroR-203 has been shown to target antiapoptotic protein bcl-w to increase cell survival [159], target survivin to cause G1 cell cycle arrest [160] and target LIM and SH3 protein 1 to inhibit migration and invasion [161]. MicroR-203 can also target p63, which maintains the balance of epithelial cell proliferation and differentiation. E7 decreases miRNA-205 via pRb [162]. MicroRNA-205 was considered to be a tumor suppressor [163] but has recently been demonstrated to increase cell proliferation via E2F1 [164]. Lower miRNA205 has been associated with poor prognosis of HPV-associated head and neck cancer [165]. Xie et al. showed that miR-205 expression was frequently higher in human cervical cancer [166]. In vitro experiments demonstrated that miR-205 promoted cell proliferation and migration in human cervical cancer cells. Two miR-205 targets, cysteine rich 61 (CYR61) and connective tissue growth factor (CTGF), were identified. Consistently, both CYR61 and CTGF were downregulated in cervical cancer tissues [166]. Both CYR61 and CTGF belong to CYR61/CTGF/ nephroblastoma family of growth regulators and have both carcinogenic or tumor suppressor effectdependent tissue types. Overexpression of CYR61 in non-small cell lung cancer cells resulted in decreased cell proliferation and colony formation [167]. CTGF reduced cell migration in oral cancer [168]. However, the effect of CYR61 and CTGF in cervical cancer still needs to be established. MicroR-15a, miR-15b and miR-15b have been shown to be upregulated by E7 through E2F1 and E2F3 [169,170]. The expression of these miRNAs is increased in cervical cancer samples [170,171]. These miRs have tumor suppressor activity [172,173], decreasing cyclin E1 and leading to cell cycle arrest [174]. Their upregulation may inhibit E2F1-promoted cell cycle. MicroR-15 targets E2F1. Therefore, the regulation of miRs by E7 could play complicated effects in HPV-caused carcinogenesis. Copyright © 2015 John Wiley & Sons, Ltd.

33 HUMAN PAPILLOMAVIRUS E5 PROTEIN E5, a small hydrophobic protein, is also an oncogene of HPVs but much less studied compared with E6 and E7. E5 is not integrated into host genomes but is expressed by episomes. In an animal model, E5 alone can also cause cancer. In addition, E5 can increase the carcinogenic effects of E6 and E7 [175]. Some signaling pathways activated by E5 have been revealed (Figure 6). The activation of signaling pathways by E5 is complementary or overlapping with E6-mediated and E7-mediated pathways.

Epidermal growth factor receptor Epidermal growth factor receptor is a tyrosine kinase, which consists of an extracellular ligandbinding domain, a transmembrane region and a transduction module consisting of a tyrosine motif and several autophosphorylation sites [176,177]. EGFR is activated by multiple ligands such as epidermal growth factor (EGF), epiregulin and TGFα. EGF/EGFR is a well-known survival pathway in many cancers. It can increase cell proliferation and decrease cell apoptosis via its downstream target proteins including Akt, MAPK and COX-2 [178]. EGFR and Src have feedforwarded regulation to activate the activity of each other through phosphorylation [178]. E5 has been shown to increase sensitivity of EGFR to the stimulation of EGF [179–181]. Changes of downstream effectors have also been investigated. E5 has been shown to increase VEGF expression via activation of Akt and MAPK [182]. Another study has shown that it also activates MAPK independent of EGFR [183]. Activation of Akt can also block Bax-caused apoptosis [182].

Fas/FasL E5 has been demonstrated to inhibit the extrinsic pathway [184]. The pathway is also called cell death pathway in which specific death receptors (Drs) are activated by their corresponding ligands, for examples tumor necrosis factor (TNF) receptor 1 or 2 (TNF R1 and TNF R2) by TNF-alpha, Fas receptor by Fas ligand, DR1, DR2 by TNF-related apoptosis-inducing ligand and TNF-like weak inducer of apoptosis by Fas-associated death domain-like interleukin-1 beta-converting enzyme [185]. The extrinsic pathway is initiated by FasL, which binds to Fas receptor, leading to activation of DRs, causing caspase 8 activation and apoptosis. Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

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Figure 6. E5-induced pathways. E5 can sensitize epidermal growth factor receptor (EGFR) to epidermal growth factor stimulation, leading to increased activation of EGFR downstream pathways phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK). Akt can upregulate vascular endothelial growth factor (VEGF) to increase angiogenesis and block Bax to decrease apoptosis. E5 can also block the Bap31-induced Fas/FasL apoptotic pathway

E5 can inhibit this pathway via various mechanisms. In HaCaT cells, E5 expression reduced FasLstimulated Fas by twofold and subsequently inhibited caspase 8 activation [186,187]. TNF-related apoptosisinducing ligand-induced cell death was also inhibited by E5. The mechanism could be the binding of E5 to Bap31 protein, which is supported as follows: (1) E5 and Bap31 can be coimmunoprecipitated; (2) they are colocalized in the endoplasmic reticulum; and (3) deletion of C terminus of E5 results in the loss of the interaction [188]. When Bap31 is cleaved into p20Bap31, it promotes cell apoptosis through the DR pathway, and binding of E5 could prevent such cleavage [189]. A recent report demonstrated that E5 could activate A4, a small transmembrane lipoprotein, which in turn increased cell proliferation. A4 is known to interact with Bap31 [190]. THE CONSEQUENCES OF SIGNALING ALTERATIONS IN HUMAN PAPILLOMAVIRUS-INFECTED CELLS

Cell proliferation Cancer is defined as abnormal cell growth characterized by increased cell proliferation and decreased apoptosis. Abnormally increased cell proliferation is one of the main characteristics of cancer [191]. Although not all hyperproliferations cause cancer, increased cell proliferation could Copyright © 2015 John Wiley & Sons, Ltd.

facilitate accumulation of mutated genes necessary for carcinogenesis. Cell proliferation is caused by cell growth and division controlled by cell cycle. Cell cycle proceeds in four phases as indicated in Figure 7 including G1, S, G2 and M phases [192]. Each phase of a cell cycle is regulated by a family of serine/threonine protein kinases, which are heterodimers consisting of a CDK (catalytic) and a cyclin (regulatory) [192]. E6 and E7 can affect these cyclins via various pathways to promote cell proliferation. Among them, cyclin D is most studied. E6 can activate the Akt/myc pathway and decrease p16 to increase cyclin D [53]. Increased cyclin B1 by E6 has also been reported, which promotes G2 phase [193–195]. HPV 16 E7 can increase cyclin A by direct interaction [196,197]. Silencing of E6/E7 by siRNA reduced cervical cancer cell proliferation in both in vitro and in vivo [198,199]. Debnath et al. studied co-operative effects of activation of Akt and expression of E7 or cyclin D1 in cultured cancer cells [200]. It was found that Akt activation alone or E7 expression alone was insufficient to maintain cell proliferation without growth factors. However, the combination of activation of Akt and expression of E7 markedly increased cell proliferation as indicated by cell proliferation marker Ki-67Akt downstream target mTOR that has been shown to play a key role as rapamycininhibited cell growth in E6/E7 transduced Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

Signaling pathways in HPV-associated cancers

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Figure 7. Human papillomaviruses, cell cycle and cell proliferation. A cell undergoes a cycle for proliferation with four phases, G1, S, G2 and M. Cell cycle is sophisticatedly regulated by cyclins and cyclin-dependent kinases (CDKs). E7 can block Rb/E2F to increase cyclin-E activity and thus accelerate cell cycle. E7 can also promote cyclin-A activity. E6 increases the activities of cyclins D and B

epithelial cells [201]. PDZ protein MAGI-1 has been shown to mediate the E6-induced cancer cell proliferation [39], and expression of MAGI-1 through disrupting its interaction with E6 resulted in decreased proliferation. In patients with HPV-associated cancers, cell proliferation in tumor tissues is increased. It has been shown that cell cycle proteins p16, Ki-67, cyclin D1, p53 and ProEx C are altered in 144 cervical tissue samples, showing increased proliferation [202]. Molecules related to cell proliferation have a significant increase from low-grade to high-grade lesions and cancer in HPV-associated cancers [203].

and pRb was sufficient to cause oral keratocyte immortalization [206]. Activation of PI3K/Akt by E5/E6/E7 can increase cell survival by reducing the intrinsic apoptotic pathway [207]. PDZ protein MAGI-1 has been shown to mediate the E6decreased cancer cell apoptosis [39]. Overexpression of MAGI-1 through disruption of its interaction with E6 resulted in increased apoptosis. E5 can decrease both extrinsic and intrinsic pathways (Figure 6) [182,184]. Therefore, HPVs use multiple oncoproteins, which alter multiple signaling pathways to decrease host cell apoptosis.

Genomic instability Apoptosis Another characteristic of cancer cells is decreased cell apoptosis [191]. There are two pathways involved in cell apoptosis: extrinsic, also called the death receptor pathway, and intrinsic, also called the mitochondrial pathway. E5/E6/E7 can decrease apoptosis through both apoptotic pathways via altering multiple signaling pathways. Targeting HPV E6 with peptide aptamers caused cancer cell apoptosis [204]. Qi et al. showed that simultaneous silencing of HPV18 E6 and E7 induced apoptosis in HeLa cells, indicating an important role for these oncoproteins in maintaining cancer cell survival [205]. E6 inhibition of p53 can greatly reduce intrinsic apoptosis to reduce DNA damage-initiated apoptosis. Smeets et al. showed that inactivation of p53 Copyright © 2015 John Wiley & Sons, Ltd.

Genomic instability plays a key role in carcinogenesis. It promotes gene mutations, which in turn affect cell proliferation and apoptosis. HPVE5, E6 and E7 could increase genomic instability via several mechanisms. Akt activation can cause genomic instability [208]. Alteration of the pRb/E2F1 pathway by E7 also leads to genomic instability [209]. Inactivation of p53 decreases DNA damage-induced cell death and increases tolerance to genomic instability. HPV-caused genomic instability is initiated by abnormal amplification of centrosomes [5]. Centrosomes are microtubule-organizing centers, consisting of a pair of centrioles and pericentriolar proteins [210]. Centrosomes are important regulators of cell division to ensure a cell is divided into two [210]. In normal conditions, centrosome duplicates once prior to mitosis to form two spindle Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

36 poles. The duplication begins in late M phase/early G1 phase. HPV E7 causes rapid induction of centrosomes with more than two copies, leading to abnormal cell division and genomic instability [211,212]. The pathway is mediated by CDK2, which is activated by Akt [213,214]. Increased CDK2 activity in turn promotes polo-like kinase 4, a rate-limiting enzyme in centriole replication [215]. It is known that deletion of polo-like kinase 4 causes defects in centriole replication, while overexpression results in multiple centrioles [215]. Genomic instability can cause accumulation of gene mutations. In cervical cancer, many gene mutations have been reported with several mutations involved in the PI3K/Akt pathway including PIK3CA, PTEN and EGFR and Ras [216–220]. These mutations can further activate the PI3K/Akt pathway in cervical cancer. A report showed that PIK3CA mutated at 31.3%, Kirsten rat sarcoma viral oncogene homologue at 8.8% and EGFR at 3.8% of cervical cancers [218]. The list of gene mutations in HPV-associated cervical cancer has been expanded in a recent study including recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%) and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%) [221]. This study also confirmed previously reported mutations such as PIK3CA (14%), PTEN (6%) and STK11 (5%). Therefore, HPV oncogenes initiate signaling pathway alterations, which cause genomic instability, leading to accumulation of gene mutations, which accelerate changes of signaling pathways necessary for carcinogenesis.

Drug resistance Drug resistance to anticancer therapeutic agents is a major reason responsible for treatment failure and high rate of cancer-related deaths [222–224]. Patients may not respond to initial therapy (inherent drug resistance) or develop drug resistance subsequently after effective initiation (acquired resistance) [222]. Multiple mechanisms for drug resistance have been elucidated including drug target mutations, increased drug detoxification system to reduce intracellular drug concentrations, increased resistance to apoptosis and abnormal activation of signaling pathways [225–232]. PI3K/Akt is well known to cause drug resistance in cancer. Activation of the pathway by insulin, IGF-1 or mutations has been associated with drug Copyright © 2015 John Wiley & Sons, Ltd.

J. Chen resistance to chemotherapeutic drug 5-fluorouracil (5-FU) and oxaliplatin [229,230]. In cervical cancer, activation of PI3K/Akt causes resistance to radiotherapy, and inhibition of the pathway increased efficacy of radiation [233]. A recent study demonstrated that activation of the PI3K/Akt pathway by E6 caused drug resistance to cisplatin in HPV-associated lung cancer mediated by the downstream target of the PI3K/Akt pathway inhibitors of anti-apoptotic protein [234]. Overexpression of hTERT increased 5-FU effect on HeLa cells to cause apoptosis [235]. Targeting these signaling pathways has been an effective strategy to overcome drug resistance in cancer treatment [236,237]. Many approaches, particularly, small molecule inhibitors, have been developed for the treatment. The combination therapy of targeted therapy with other treatment regime is detailed in the late section—therapeutic implications.

Cell mobility and metastasis Cell migration plays a key role in cancer metastasis. To be able to migrate to a new place, a cancer cell must detach from the original site, migrate to the new place, attach and grow in a new environment. Signaling pathways play key roles in the process of cancer metastasis [238]. They can control cell morphological change, cell polarity and cell mobility to meet the requirements of migration. Many signaling changes caused by HPV E6 and E7 are associated with cell morphology and migration such as the PI3K/Akt and RhoA pathways [152,157]. MicroRNAs have also been associated with cell morphology control. Therefore, HPV E6 and E7 can also facilitate cancer metastasis. Loss of PTEN has been associated with increased metastasis in patients [239]. Activation of MAPK increased metastasis of cervical cancer [240]. Antiviral treatment has been shown to have anti-metastatic effect in HPV-positive cells [241].

Cancer stem cells It has been realized that cancer cells in a tumor or in a cell line are heterogeneous, which are composed of many different phenotypic cancer cells [242,243]. One small population of cancer cells is called cancer stem cells (CSCs). These cells have the property of renewal and can differentiate to other types of cancer cells. CSCs are considered to Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

Signaling pathways in HPV-associated cancers be only cells within a tumor that can proliferate extensively and drive tumorigenic growth [242,243]. The other types of cancer cells are terminal differentiated cells, which are not renewed. CSCs have also characteristics of normal stem cells, that is slow cycling, altered DNA repair machinery and high expression levels of anti-apoptotic genes and ATPbinding cassette (ABC) transporters [244]. These characteristics render CSCs drug resistant, for example CSCs are less sensitive to cisplatin than non-CSCs in HNSCC [245]. Cancer stem cells have been identified in acute myeloid leukemia and later found in other cancer types as well, for example breast cancer, brain cancer, colon cancer, neck and head cancer, pancreas cancer, liver cancer and melanoma. In cervical cancer, CSCs have been isolated by various biomarkers including cell surface markers, Hoechst staining and formation of spheres. Qi et al. isolated sphere cells from cervical cancer cell line HeLa [246]. These stem cells account for only about 1% of total population. They are small, round cells with increased biomarkers Oct3/4, CD133 and breast cancer resistance protein. However, aldehyde dehydrogenase is not changed. Stem cells isolated have been shown to have increased drug resistance to chemotherapeutic agent Trichostatin and radiation treatment. CSCs have also been isolated from two other cervical cell lines SiHa and CaLo by their increased ability to efflux Hoechst 33342 [247]. These cells had increased ABCG2 and ABCB1. Not many studies have been performed for the role of HPV in CSCs. It has been compared with HPV-positive HNSCC cancers with HPV-negative ones for the amount of CSCs contained. Zhang et al. found that HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC [248], while Tang et al. showed that the proportion of CSC was not significantly different in HPV-positive or HPV-negative HNSCC cell lines [245]. These experiments may not be able to provide valuable evidence for the role of HPV in CSCs because other cancers can be caused by risk factors, which also affect CSC abundance. Transduction of HPV negative cancer cells with HPV E6/E7 can reflect the role of E6/E7 in CSCs. It has been shown to increase colony formation that was observed in both CSCs and non-CSCs. However, its effect on stemness is not well studied. This is an important area, where detailed studies are warranted. Copyright © 2015 John Wiley & Sons, Ltd.

37 THERAPEUTIC IMPLICATIONS HPV-associated cancers that cannot be removed by surgery can be treated by multiple approaches such as chemotherapeutic agents, antiviral therapy, immunotherapy, radiotherapy, phytochemicals and targeted therapy (Figure 8).

Chemotherapy A number of chemotherapeutic agents have been used in cervical cancer. Cisplatin has been shown to be most effective among all drugs tested including cisplatin, carboplatin, 5-FU, cyclophosphamide, chlorambucil, melphalan, methotrexate, vincristine, bleomtcin, adriamtcin, mitomycin C, ifosfamimde, pclitaxel, irinotecan, gemcitabine, vinorelbine, docetaxel, doxorubicin and mitolactol. However, cisplatin only has 10–20% of responses [249]. An increase of dosage of cisplatin resulted in severe renal toxicity. Carboplatin is a derivative of cisplatin but with reduced renal toxicity. Overall, single chemotherapeutic agents can only produce low efficacy, which can only extend patients’ lives to 12 months and thus is regarded as a palliative therapy [250]. As single agents are not efficient, various combination therapies have been applied. Chemotherapeutic agents have been used for double or triplet combination therapy to increase therapeutic effectiveness. For example, cisplatin has been used together with 5-FU, and the combination therapy showed various responses from 22% to 58% [251– 253]. Cisplatin and capecitabine together produced responses of 30–50% [254,255]. However, the median survival was not improved. The use of chemotherapeutic agents usually results in drug resistance caused by activation of signaling pathways. Combination of chemotherapeutic agents and targeted therapy could be more effective, as activation of signaling pathways is one of the major reasons for drug resistance of chemotherapeutic agents. For example, 5-FU can stimulate the Src and PI3K/Akt pathways, resulting in drug resistance and inhibition of Src that increased the efficacy of 5-FU markedly [256].

Antiviral and anti-oncogene therapies Because expression of HPV oncogenes through episomes or integrated E6 and E7 genes is important for the maintenance of cancer cells, elimination or inhibition of HPVs, HPV oncogenes has been studied for the treatment of HPV-associated cancers. Several antiviral agents have been tested. Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

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Figure 8. Therapy strategy against human papillomavirus (HPV)-associated cancers. HPV oncogenes E5, E6 and E7 activates cellular signaling pathways to promote cancer cell survival. Treatment strategy can target the process in different levels including decreasing HPVs and their oncogene expression by short hairpin RNAs (shRNAs) or small molecule inhibitors, targeting cellular signaling pathways by inhibitors or phytochemicals and killing cancer cells by chemotherapy or radiotherapy or immunotherapy

Cidofovir has been shown to restore p53 and increase radiosensitivity in HPV-associated cancers [257]. RNA interference has been used to inhibit expression of E6 and E7 [258]. Transduction of lentiviral vectors delivered shRNA against E6/E7 into HeLa cells caused apoptosis [259–262]. Combination of shRNA against both E6 and VEGF was more effective for the treatment of cervical cancer than single shRNA or combination therapy with chemotherapeutic drugs [262,263]. It has been shown that siRNA against E6 increased the sensitivity of SiHa cells to cisplatin [264]. In a xenograft model of cervical cancer, injection of shRNA against E6/E7 reduced tumor growth [265]. A range of small molecule inhibitors have been developed to inhibit E6 and E7 or their binding to partner proteins [266].

Targeted therapy As E6/E7 affects cancer via signaling pathways, modulation of these signaling pathways has therapeutic implications. As multiple signaling pathways have been altered by E6 and E7, it is possible to use multiple small chemicals to reverse Copyright © 2015 John Wiley & Sons, Ltd.

these pathways such as reactivation of p53, inhibition of the PI3K/Akt pathway, inhibition of the Notch pathway and supplementation of decreased miRNAs. Among them, targeted therapies against the EGFR, VEGF, Src, PI3K/Akt and Erk pathways have been the best studied to date (Figure 9).

Anti-phosphoinositide 3-kinase/protein kinase B Activation of the PI3K/Akt pathway has been associated with increased cancer cell proliferation, decreased apoptosis, increased cell migration and decreased drug sensitivity in many cancers including cervical cancer [41,45,229,267,268]. Targeted therapy against PI3K/Akt has been used in many cancers [236,269]. Noh et al. showed that activation of Akt by HPV 16 E7 was responsible for cancer cells to escape immune responses [270]. Therefore, targeting PI3K/Akt may be an effective approach for the treatment of HPVassociated cancer. Several studies have tested the effect of PI3K/Akt pathway inhibitors on cervical cell lines. Rashmi et al. used two allosteric Akt inhibitors Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

Signaling pathways in HPV-associated cancers (SC-66 and MK-2206) in combination with the glucose analog 2-deoxyglucose in C33A cells, which have activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations [271]. Results showed that inhibition of the PI3K/Akt by these inhibitors decreased cell viability through a non-apoptotic mechanism. 2-Deoxyglucose further increased cell viability. SC-66 also inhibited cell migration. Xia et al. tested the effect of PI3K/Akt/COX-2 pathway inhibitors Ly294002 and celecoxib on the sensitivity of radiotherapy in HeLa cells and found that the combination of Ly294002, celecoxib and radiation produced treatment effects [233]. The combination of inhibition of PI3K/Akt and radiotherapy has also been tested in an animal model of cervical cancer [272,273]. In BALB/C nude mice with xenografted HeLa cells, LY294002 decreased cell survival and xenograft tumor growth. However, the combination of LY294002 and radiation resulted in synergistic reduction of tumor growth [273].

Anti-mammalian target of rapamycin Inhibitors of PI3K/Akt downstream signaling molecule mTOR have also been developed. These inhibitors have been tested in cervical cancer cell lines and animal models. Among them,

39 temsirolimus has been used in a phase-II clinical trial. It increased cervical cancer patient survival time, that is 3% partial response, and 57.6% had stable disease [274]. Coppock et al. established a mouse cancer cell line by transducing both E6/E7 oncogenes and mutated H-Rasv12 gene into mouse oropharyngeal epithelial cells for the test of the effect of rapamycin [201]. The cells can grow into tumor in a xenograft model. Rapamycin has been used to treat the mice in combination with cisplatin, decreasing cancer cell proliferation and prolonging long-term survival rate of mice with the xenograft tumors.

Anti-vascular endothelial growth factor Human papillomavirus oncogenes cause upregulation of VEGF, which can increase cancer cell survival and angiogenesis. In cervical cancer, VEGF is overexpressed. Therefore, anti-VEGF has been used in clinical trial for the treatment of cervical cancer. Bevacizumab, a monoantibody against VEGF, is a most-studied agent for anti-VEGF therapy. A recent study showed that addition of bevazumab to chemotherapeutic cisplatin– paclitaxel or topotecan–paclitaxel regime increased treatment efficacy, increasing response rate from 36% to 48% and overall survival time from 13.3 months to 17.0 months [275]. It can also increase

Figure 9. Targeted therapy against key signaling pathways in human papillomavirus-associated cancers. Many small molecule inhibitors have been developed to target key signaling molecules in human papillomavirus-associated cancers including epidermal growth factor receptor (EGFR), extracellular signal-regulated kinases (ERK), vascular endothelial growth factor (VEGF), Src and mammalian target of rapamycin (mTOR). Dual inhibitors against both phosphoinositide 3-kinase (PI3K) and mTOR have also been developed. Akt, protein kinase B; GSK, glycogen synthase kinase


Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv

40 treatment efficacy of radiation and cisplatin alone [276]. Many small molecule inhibitors of VEGF have also been developed and tested in clinical trials with benefit but limited improvement of survival time.

Anti-epidermal growth factor receptor Epidermal growth factor receptor gene amplification has been associated with poorer outcome of cervical cancer, and thus, anti-EGFR has been emphasized in the treatment of cervical cancer [277]. Both antibody against EGFR (cetuximab) and small molecule inhibitors of EGFR (gefitinib and erlotinib) have been tested. Cetuximab produced synergistic effects with antiviral agent cidofovir in HeLa and Me180 cells in both in vitro cell cultures and in vivo xenograft model [278]. Cetuximab has also been tested in a clinical trial with platinum and 5-FU in 121 head and neck cancer patients and extended overall survival time to 11 months [279]. Another phase-II clinical trial with combination of cetuximab and cisplatin showed that the regime was well tolerated but cetuximab had no further benefit than cisplatin alone [280]. Gefitinib also resulted in stable disease in 20% of patients of recurrent cervical cancer in a clinical trial [281]. Clinical trials of erlotinib showed safety [282] but limited benefit even in combination with topotecan [283] and cisplatin [284].

J. Chen D1. EGCG has been demonstrated to increase the sensitivity to cisplatin [285]. Curcumin has also been shown to suppress HPV oncogene expression and thus increase p53 and pRb [290]. Singh et al. showed that curcumin decreased estrodiol-induced cervical cancer cell proliferation and caused apoptosis [291] Curcumin can decrease the levels of E7, proliferating cell nuclear antigen and cyclin D1 elevated by estrogen but not E6, telomerase and p16. Curcumin overcame drug resistance to cisplatin in SiHa cells by inhibiting metalloprotease 1, P-glycoprotein, cyclin D1 and upregulation of p53, peripheral benzodiazepine receptor, p21 and p27 [292]. Nanoparticlepacked curcumin increased the sensitivity of cervical cancer to chemotherapeutic agent paclitaxel through inhibition of the Akt pathway [293]. In a xenograft model using CaSki cells, curcumin reduced tumor size by decreasing VEGF, COX-2 and EGFR [294]. A cream containing curcumin eliminated HPV+ cells in mice [295]. Quercetin has also caused HeLa apoptosis and cell cycle arrest in G2/M phase [296,297]. It was shown to increase expression of p53 and p21 and decrease cyclin D1. The mitochondrial apoptotic pathway was activated with upregulation of proapoptotic bcl-2 family members and cytochrome c and Apaf-1 and caspases and downregulation of bcl-2 and survivin. Quercetin also inhibited NF-κB [296].

Applications of phytochemicals Phytochemicals could be used for the treatment of cancer because of their ability to inhibit multiple signaling pathways. Some phytochemicals have been well studied in cervical cancer such as epigallocatechin gallate (EGCG), resveratrol and curcumin and quercetin. EGCG is a major green tea component, which decreases cell proliferation and increases drug sensitivity in cervical cancer [285,286]. The mechanisms are inhibition of several key molecules of signaling pathways. In Caski, EGCG induced G1 arrest and apoptosis [287,288]. Treatment of HeLa cells with EGCG induced dose-dependent and time-dependent inhibitions of proliferation and increased apoptosis [289]. EGCG activated the mitochondrial apoptotic pathway indicated by increases of reactive oxygen species, Bax/Bcl-2 ratio, cytochrome-c release, cleavage of procaspase-3 and -9 and poly(ADP-ribose)-polymerase. EGCG also inhibited the activity of Akt and NF-κB and reduced expression levels of cyclin Copyright © 2015 John Wiley & Sons, Ltd.

Immunotherapy An effective immune system is necessary for the eradication of cancer cells, which have been weakened by chemotherapy or targeted therapy. Although infection of HPV can elicit immune responses against virus and can clear virus in most cases, HPV oncogenes E5, E6 and E7 can inhibit immune responses to escape the clearance. E5 reduces HLA-1 expression by interacting with HLA-1 heavy chain [298]. E7 can reduce STAT-1 and decrease transporter associated with antigen processing/interferon regulatory factor and thus reduce HLA class 1 [299]. Therefore, HPV antigen presentation is reduced, and immune responses against HPV-positive cells are decreased. Various immunotherapies have been tried in cervical cancer and vulval intraepithelial neoplasia (VIN) to strengthen the immune responses to eliminate virus or against cancer cells. Imiquimod, a toll-like receptor agonist, has been tested to activate Rev. Med. Virol. 2015; 25: 24–53. DOI: 10.1002/rmv


41

innate immune cells such as dendritic cells, monocytes and macrophages in HPV-associated cancers. Imiquimod increased HPV clearance and reduced tumor size in a clinical trial with 59 cervical cancer patients [300]. The mechanism was shown to be the normalization of immune cell counts [301] and increased CD8+ cell recognition to E7 immunization [302]. Topical application of 5% imiquimod cream in 62 VIN patients resulted in 47 complete responses and 19 partial responses [303]. The effect of imiquimod was time-dependent, and patients had complete regression rate 32% (6 out of 19) at week 10, 58% (11 out of 19) at week 20 and 63% (12 out of 19) at week 52 [304]. E6 and E7 proteins have been used to elicit specific immune response to HPV-positive cells. The test in 20 patients with VIN showed partial efficacy with increased CD4 and CD8 T-cell responses [305]. E7 has been carried by a naked DNA vector for its expression in cells to increase MHC processing, resulting in increased immunogenicity of E7 and overcoming immune tolerance [306]. Imiquimod increased the effect of this DNA vaccine [307]. Cytokines have also been used to activate immune system in treating cervical cancer. The most commonly used cytokines are IL-2, IL-12, granulocyte-macrophage-CSF and IFN-alpha. Systemic application of cytokines is highly toxic, but local application is well tolerated [308].

increased activator protein 1, NF-κB and MAPK [316]. More detailed studies are needed to elucidate the role of estrogen in the carcinogenesis of cervical cancer. Nevertheless, ERα has been demonstrated to play an important role in cervical cancer in animal experiment, and selective estrogen receptor modulators raloxifene can decrease tumor growth [317,318]. In addition to inhibiting ERα, activation of ERβ has also been tested for the treatment of cervical cancer. Genistein, a phytoestrogen, which stimulates ERβ, induced apoptosis of HeLa cells [319] and sensitized chemotherapeutic agents cisplatin and camptothecins [320,321]. Geistein was shown to inhibit the PI3K/Akt, NF-κB, MAPK and MMP-9 pathways and activate apoptotic pathways [321–324]. Antiestrogen phytochemical indole-3-carbinol was shown to prevent incidence of cervical cancer in a transgenic model [325]; 19 out of 25 mice developed cervical cancer in control group, while only 2 out of 24 had cancer in indole-3-carbinol supplemented group. In cervical cancer cell lines, indole3-carbinol was shown to cause apoptosis through the intrinsic pathway indicated by the reduction of Bcl-2 protein [326]. Indole-3-carbinol also prevented PTEN loss [327] and decreased cyclin-E levels [328].

Antiestrogen therapy

Targeting cancer stem cells

Estrogen plays a key role in several cancers such as endometrial, prostate, colon and breast cancers. Epidemiological evidence has shown that estrogen is also important in cervical cancer [309]. In an animal model, estrogen has been shown to have cooperating effect with HPV 16 oncogenes E6 and E7 [310–312]. There are two types of estrogen receptors—estrogen receptor-alpha (ERα) and estrogen receptor-beta (ERβ). The activation of these receptors has opposite effects in some cancers. In colon cancer, ERα increases carcinogenesis via activation of signaling pathways Akt and MAPK, while ER-beta inhibits these pathways. Both ERα and ERβ are expressed in cervical cancer [313]. ERα is decreased, while ERβ is maintained in cervical cancer [313,314]. Interestingly, ERα expression in stromal cells is increased [314], and ERα in these cells is necessary for the development of cervical cancer [315]. The signaling pathways altered by estrogen in cervical cancer are not well studied. A recent study using microassay showed that it

It has been recognized that CSCs play key roles in cancer treatments. Conventional therapy can result in decreased differentiated cancer cells while CSCs are resistant to the therapy. The therapy could eliminate differentiated cancer cells, leading to initial shrinkage of tumor. However, remained CSCs will continue to produce differentiated cancer cells, causing relapse of cancer. Therefore, elimination of CSCs could lead to curable cancer. Signaling pathways are important in maintaining CSCs, such as the PI3K/Akt and MAPK pathways. Akt activation has been associated with stemness in various cancers such as pancreatic cancer, ovarian cancer, breast cancer and glioma [329–332]. As stem cells are drug resistant, increased stemness may account for increased drug resistance. Inhibition of PI3K/Akt pathway caused a decreased sphere formation in cancer cell populations in breast cancer, glioma and prostate cancer [333–335]. Studies have also shown that IGF-1 is associated with colon



42

J. Chen

CSCs [336]. Activation of the PI3K/Akt pathway decreases the sensitivity of CSCs to chemotherapeutic drugs [335,337]. The downstream of the Akt pathway such as GSK/β-catenin has been demonstrated to be of importance in CSCs [330]. Thus, targeting signaling pathways may eradicate CSCs. This has been demonstrated by a recent breakthrough by inhibiting signaling molecule BMI-1 to reduce colon CSCs’ renewal ability [338]. The approach may be applicable in HPV-associated cancer. A recent study showed that CD200, a membrane protein, was overexpressed in HPV-positive HNSCC cell lines and the overexpression of CD200 was associated with increased BMI-1 [339]. In vivo, CD200 increased resistance to chemoradiation, indicating the importance of BMI-1 in HPV-associated cancers, and inhibition of BMI-1 may have similar effect as in colon cancer. In summary, various approaches have been used for the treatment of HPV-associated cancers, especially cervical cancer. However, the single treatment approach has very limited effect. Combination therapy may be promising, particularly the combination of targeted therapy with other approaches. Single inhibition of a signaling molecule in cervical cancer could be not effective. This may be due to the activation of multiple signaling pathways by HPV oncogenes E5, E6 and E7.

Simultaneous inhibition of two or more key signaling molecules may produce much better effects. At present, the inhibition of multiple molecules in HPV-associated cancers has not been optimized. CONCLUSIONS HPV oncogenes E5, E6 and E7 play key roles in HPV-associated cancer by activating multiple cancer survival signaling pathways and inhibiting cancer suppressor proteins. Altered signaling pathways in turn promote cell proliferation, decrease cell apoptosis and increase cell migration and drug resistance. The effects of E5, E6 and E7 could be synergistic. Targeting E5-mediated, E6mediated and E7-mediated signaling pathways is of therapeutic value. Among them, the PI3K/Akt pathway activated by E5, E6 and E7 as well as mutations of genes that encode elements of the pathway could be inhibited, which may provide an effective approach for the treatment of HPVassociated cancer. Although anti-EGFR and antiVEGF have been used in clinical trials in cervical cancer, outcomes are not satisfactory. As multiple signaling pathways are activated by HPV oncogenes, it may be necessary to inhibit multiple signaling molecules for the treatment of HPVassociated cancers.

REFERENCES 1. Bouvard V, Baan R, Straif K, et al. A review of human carcinogens—Part B: biological agents. The Lancet Oncology 2009; 10: 321–322. 2. Nobre RJ, Herráez-Hernández E, Fei J-W,

transformation. Nature Reviews Cancer 2010; 10: 550–560.

carcinoma than the prototype. Cancer Research 1998; 58: 829–833.

6. Zhao KN, Chen J. Codon usage roles in

10. DeFilippis RA, Goodwin EC, Wu L,

human papillomavirus. Reviews in Medical

DiMaio D. Endogenous human papillo-

Virology 2011; 21: 397–411.

mavirus E6 and E7 proteins differentially

et al. E7 oncoprotein of novel human pap-

7. Bible JM, Mant C, Best JM, et al. Cervical

regulate proliferation, senescence, and

illomavirus type 108 lacking the E6 gene

lesions are associated with human papillo-

apoptosis in HeLa cervical carcinoma

induces

organotypic

mavirus type 16 intratypic variants that

cells.

keratinocyte cultures. Journal of Virology

have high transcriptional activity and in-

1551–1563.

2009; 83: 2907–2916.

creased usage of common mammalian co-

11. Narisawa-Saito

dysplasia

in

3. Stubenrauch F, Straub E, Fertey J, Iftner T. The E8 repression domain can replace the

Journal

of

Virology M,

2003;

Inagawa

77: Y,

dons. Journal of General Virology 2000; 81:

Yoshimatsu Y, et al. A critical role of

1517–1527.

MYC for transformation of human cells

E2 transactivation domain for growth inhi-

8. Londesborough P, Ho L, Terry G, Cuzick

bition of HeLa cells by papillomavirus E2

J, Wheeler C, Singer A. Human papillo-

proteins. International Journal of Cancer

mavirus genotype as a predictor of persis-

2007; 121: 2284–2292.

by HPV16 E6E7 and oncogenic HRAS. Carcinogenesis 2012; 33: 910–917. 12. Ocadiz-Delgado R, Castañeda-Saucedo E,

tence and development of high-grade

Indra AK, et al. RXRα deletion and

4. Münger K, Baldwin A, Edwards KM, et al.

lesions in women with minor cervical ab-

E6E7 oncogene expression are sufficient

Mechanisms of human papillomavirus-

normalities. International Journal of Cancer

to

induced oncogenesis. Journal of Virology

1996; 69: 364–368.

lesions in vivo. Cancer Letters 2012; 317:

9. Zehbe I, Wilander E, Delius H, Tommasino

2004; 78: 11451–11460. 5. Moody CA, Laimins LA. Human papillomavirus

oncoproteins:

pathways

to

induce

cervical

malignant

226–236.

M. Human papillomavirus 16 E6 variants

13. Hoe KK, Verma CS, Lane DP. Drugging

are more prevalent in invasive cervical

the p53 pathway: understanding the route



Signaling pathways in HPV-associated cancers to clinical efficacy. Nature Reviews Drug Discovery 2014; 13: 217–236.

43 24. Ben KY, Teissier S, Tan M, et al. The hu-

and triggers its degradation by the pro-

man papillomavirus E6 oncogene re-

teasome. Journal of Virology 2005; 79: 4229–4237.

14. Rufini A, Tucci P, Celardo I, Melino G. Se-

presses a cell adhesion pathway and

nescence and aging: the critical roles of

disrupts focal adhesion through degrada-

p53. Oncogene 2013; 32: 5129–5143.

tion of TAp63β upon transformation.

Human

PLoS Pathogens 2011; 7: e1002256.

oncoprotein interferes with the epithelial

15. Huibregtse J, Scheffner M, Howley P.

34. Facciuto F, Valdano MB, Marziali F, et al. papillomavirus

(HPV)-18

E6

cell polarity Par3 protein. Molecular Oncol-

Localization of the E6-AP regions that

25. Park JS, Kim EJ, Lee JY, Sin HS,

direct human papillomavirus E6 bind-

Namkoong SE, Um SJ. Functional inacti-

ing,

and

vation of p73, a homolog of p53 tumor

35. Thomas M, Narayan N, Pim D, et al. Hu-

association

with

p53,

ogy 2014; 8: 533–543.

proteins.

suppressor protein, by human papilloma-

man papillomaviruses, cervical cancer

Molecular and Cellular Biology 1993; 13:

virus E6 proteins. International Journal of

and cell polarity. Oncogene 2008; 27:

4918–4927.

Cancer 2001; 91: 822–827.

ubiquitination

of

associated

7018–7030. 36. Massimi P, Gammoh N, Thomas M, Banks

16. Brimer N, Lyons C, Vande Pol SB. Associ-

26. Maas A-M, Bretz AC, Mack E, Stiewe T.

ation of E6AP (UBE3A) with human papil-

Targeting p73 in cancer. Cancer Letters

L. HPV E6 specifically targets different cel-

lomavirus type 11 E6 protein. Virology

2013; 332: 229–236.

lular pools of its PDZ domain-containing

27. Moll UM, Slade N. p63 and p73: roles in

2007; 358: 303–310. 17. Ansari T, Brimer N, Pol SBV. Peptide in-

development and tumor formation 11 Na-

teractions stabilize and restructure hu-

tional Cancer Institute. Molecular Cancer

man

Research 2004; 2: 371–386.

papillomavirus

type

16

E6

to

interact with p53. Journal of Virology

tumour

suppressor

substrates

for

proteasome-mediated degradation. Oncogene 2004; 23: 8033–8039. 37. Adey NB, Huang L, Ormonde PA, et al.

28. Ye F, Zhang M, Aregger M, et al. Struc-

Threonine

phosphorylation

of

the

tures and target recognition modes of

MMAC1/PTEN PDZ binding domain

18. Scheffner M, Huibregtse JM, Vierstra RD,

PDZ domains: recurring themes and

both inhibits and stimulates PDZ binding.

Howley PM. The HPV-16 E6 and E6-AP

emerging pictures. Biochemical Journal

complex functions as an ubiquitin-protein

2013; 455: 1–14

2012; 86: 11386–11391.

ligase in the ubiquitination of p53. Cell 1993; 75: 495–505. 19. Scheffner M, Werness BA, Huibregtse JM, Levine

AJ,

Howley

PM.

The

E6

29. Vanitha KS, Christian K, Miranda T, Law-

Tsuchida K, Kimmelman AC, Chan AM.

rence B. PDZ domains: the building blocks

Regulation of PTEN binding to MAGI-2

regulating tumorigenesis. Biochemical Jour-

by two putative phosphorylation sites at

nal 2011; 439: 195–205.

threonine 382 and 383. Cancer Research

oncoprotein encoded by human papillo-

30. Kiyono T, Hiraiwa A, Fujita M, Hayashi

mavirus types 16 and 18 promotes the

Y, Akiyama T, Ishibashi M. Binding of

Cell

Cancer Research 2000; 60: 35–37. 38. Tolkacheva T, Boddapati M, Sanfiz A,

2001; 61: 4985–4989. 39. Kranjec C, Massimi P, Banks L. Restora-

E6

tion of MAGI-1 expression in HPV posi-

oncoproteins to the human homologue

tive tumour cells induces cell growth

20. Lorenz LD, Cardona JR, Lambert PF. Inac-

of the Drosophila discs large tumor sup-

arrest and apoptosis. Journal of Virology

tivation of p53 rescues the maintenance of

pressor protein. Proceedings of the Na-

high risk HPV DNA genomes deficient in

tional

expression of E6. PLoS Pathogens 2013; 9:

11612–11616.

degradation

of

p53.

1990;

63:

1129–1136.

high-risk

human

Academy

of

papillomavirus

Sciences

1997;

94:

of

human

J,

Dienstmann

R,

Serra

V,

Tabernero J. Development of PI3K inhibi-

31. Lee SS, Weiss RS, Javier RT. Binding

e1003717. 21. Kho E-Y, Wang H-K, Banerjee NS, Broker

2014; JVI: 03247–03213. 40. Rodon

virus

oncoproteins

to

tors: lessons learned from early clinical trials. Nature Reviews. Clinical Oncology 2013; 10: 143–153.

TR, Chow LT. HPV-18 E6 mutants reveal

hDlg/SAP97, a mammalian homolog of

p53 modulation of viral DNA amplifica-

the Drosophila discs large tumor sup-

41. Chen J, Zhao K, Li R, Shao R, Chen C. Ac-

tion in organotypic cultures. Proceedings

pressor protein. Proceedings of the Na-

tivation of PI3K/Akt/MTOR pathway

of the National Academy of Sciences 2013;

tional Academy of Sciences 1997; 94:

and dual inhibitors of PI3K and MTOR in

110: 7542–7549.

6670–6675.

endometrial

cancer.

Current

Medicinal

Chemistry 2014; 21: 3070–3080.

22. Massimi P, Shai A, Lambert P, Banks L.

32. Lee C, Laimins LA. Role of the PDZ

HPV E6 degradation of p53 and PDZ con-

domain-binding motif of the oncoprotein

42. Polivka Jr J, Janku F. Molecular targets for

taining substrates in an E6AP null back-

E6 in the pathogenesis of human papillo-

cancer therapy in the PI3K/AKT/mTOR

ground. Oncogene 2008; 27: 1800–1804.

mavirus type 31. Journal of Virology 2004;

pathway. Pharmacology & Therapeutics

23. Mesplède

T,

Gagnon

D,

Bergeron-

78: 12366–12377.

2014; 142: 164–175.

Labrecque F, et al. p53 degradation activ-

33. Favre-Bonvin A, Reynaud C, Kretz-Remy

43. Zhang X, Li X-r, Zhang J. Current status

ity, expression, and subcellular localiza-

C, Jalinot P. Human papillomavirus type

and future perspectives of PI3K and

tion of E6 proteins from 29 human

18 E6 protein binds the cellular PDZ pro-

mTOR inhibitor as anticancer drugs in

papillomavirus genotypes. Journal of Virol-

tein TIP-2/GIPC, which is involved in

breast cancer. Current Cancer Drug Targets

ogy 2012; 86: 94–107.

transforming growth factor β signaling

2013; 13: 175–187.



44

J. Chen

44. Grunt W T, Mariani L, Novel G. Ap-

and Mad during epidermal differentiation

proaches for molecular targeted therapy

and HPV-associated tumorigenesis. Onco-

of

gene 1995; 11: 2487–2501.

breast

cancer:

interfering

with

PI3K/AKT/mTOR signaling. Current Cancer Drug Targets 2013; 13: 188–204. 45. Chen J. Multiple signal pathways in

55. Dellas A, Schultheiss E, Leivas M, Moch

β-catenin antisense treatment decreases β-

H, Torhorst J. Association of p27Kip1, cy-

catenin expression and tumor growth rate

clin E and c-myc expression with progres-

in colon carcinoma xenografts. Journal of

sion

2011; 12: 1063–1070.

cervical neoplasms. Anticancer Research

XF,

Chen

JZ.

Obesity,

the

PI3K/Akt signal pathway and colon Obesity

cancer.

Reviews

2009;

10:

610–616. 47. Contreras-Paredes Hernández

E,

A,

De

la

Cruz-

Martínez-Ramírez

I,

Dueñas-González A, Lizano M. E6 vari-

Cancer Research 2000; 60: 4761–4766. 67. Green DW, Roh H, Pippin JA, Drebin JA.

obesity-associated cancer. Obesity Reviews 46. Huang

complex in early colorectal carcinogenesis.

and

prognosis

in

HPV-positive

1997; 18: 3991–3998.

Surgical Research 2001; 101: 16–20. 68. Yan D, Wiesmann M, Rohan M, et al. Elevated expression of axin2 and hnkd

56. McMurray H, McCance D. Human papil-

mRNA provides evidence that Wnt/β-

lomavirus type 16 E6 activates TERT

catenin signaling is activated in human

gene transcription through induction of

colon tumors. Proceedings of the National

c-Myc and release of USF-mediated re-

Academy

pression. Journal of Virology 2003; 77:

14973–14978.

9852–9861.

of

Sciences

2001;

98:

69. Baarsma HA, Königshoff M, Gosens R.

57. Veldman T, Liu X, Yuan H, Schlegel R. Hu-

The Wnt signaling pathway from ligand

kinase

man papillomavirus E6 and Myc proteins

secretion to gene transcription: molecular

3-kinase

associate in vivo and bind to and coopera-

mechanisms and pharmacological targets.

(akt/PI3K) signaling pathway. Virology

tively activate the telomerase reverse tran-

Pharmacology & Therapeutics 2013; 138:

2009; 383: 78–85.

scriptase promoter. Proceedings of the

ants of human papillomavirus 18 differentially

modulate

the

protein

B/phosphatidylinositol

48. Lu Z, Hu X, Li Y, et al. Human papillomavirus 16 E6 oncoprotein interferences with

National Academy of Sciences 2003; 100: 8211–8216.

66–83. 70. Pereira-Suárez AL, Meraz MA, Lizano M, et al. Frequent alterations of the β-catenin protein in cancer of the uterine cervix. Tu-

insulin signaling pathway by binding to

58. Gross-Mesilaty S, Reinstein E, Bercovich B,

tuberin. Journal of Biological Chemistry

et al. Basal and human papillomavirus E6

2004; 279: 35664–35670.

oncoprotein-induced degradation of Myc

71. Fadare O, Reddy H, Wang J, Hileeto D,

49. Zheng L, Ding H, Lu Z, et al. E3 ubiquitin

proteins by the ubiquitin pathway. Pro-

Schwartz PE, Zheng W. E-cadherin and β-

ligase E6AP-mediated TSC2 turnover in

ceedings of the National Academy of Sciences

catenin expression in early stage cervical

the presence and absence of HPV16 E6.

1998; 95: 8058–8063.

carcinoma: a tissue microarray study of

Genes to Cells 2008; 13: 285–294.

59. Mantovani F, Banks L. The human papillo-

50. Spangle JM, Münger K. The human papil-

mavirus E6 protein and its contribution to

lomavirus type 16 E6 oncoprotein acti-

malignant progression. Oncogene 2001; 20:

vates mTORC1 signaling and increases protein synthesis. Journal of Virology 2010; 84: 9398–9407. 51. Spangle JM, Munger K. The HPV16 E6

7874–7887.

mor Biology 2002; 23: 45–53.

147 cases. World Journal of Surgical Oncology 2005; 3: 38. 72. Shinohara A, Yokoyama Y, Wan X, et al. Cytoplasmic/nuclear expression without

60. Reis M, Liebner S. Wnt signaling in the

mutation of exon 3 of the β-catenin gene

vasculature. Experimental Cell Research

is frequent in the development of the neo-

2013; 319: 1317–1323.

plasm of the uterine cervix. Gynecologic Oncology 2001; 82: 450–455.

oncoprotein causes prolonged receptor

61. Clevers H, Nusse R. Wnt/β-catenin signal-

protein tyrosine kinase signaling and en-

ing and disease. Cell 2012; 149: 1192–1205.

73. Rampias T, Boutati E, Pectasides E, et al.

hances internalization of phosphorylated

62. Moon RT, Kohn AD, De Ferrari GV,

Activation of Wnt signaling pathway by

receptor species. PLoS Pathogens 2013; 9:

Kaykas A. Wnt and β-catenin signalling:

human papillomavirus E6 and E7 onco-

e1003237.

diseases and therapies. Nature Reviews Ge-

genes in HPV16-positive oropharyngeal

netics 2004; 5: 691–701.

squamous carcinoma cells. Molecular Can-

52. Boon SS, Banks L. High-risk human papillomavirus E6 oncoproteins interact with 14-3-3ζ

in

a

PDZ

binding

motif-

dependent manner. Journal of Virology 2013; 87: 1586–1595. 53. Kim SH, Koo BS, Kang S, et al. HPV inte-

63. Klaus A, Birchmeier W. Wnt signalling and its impact on development and cancer.

cer Research 2010; 8: 433–443. 74. Lichtig H, Gilboa DA, Jackman A, et al.

Nature Reviews Cancer 2008; 8: 387–398.

HPV16 E6 augments Wnt signaling in an

64. He T-C, Sparks AB, Rago C, et al. Identifi-

E6AP-dependent manner. Virology 2010;

cation of c-MYC as a target of the APC

396: 47–58. 75. Bonilla-Delgado J, Bulut G, Liu X, et al. The

gration begins in the tonsillar crypt and

pathway. Science 1998; 281: 1509–1512.

leads to the alteration of p16, EGFR and

65. Tetsu O, McCormick F. β-catenin regulates

E6 oncoprotein from HPV16 enhances the

c-myc during tumor formation. Interna-

expression of cyclin D1 in colon carcinoma

canonical Wnt/β-catenin pathway in skin

tional

cells. Nature 1999; 398: 422–426.

epidermis in vivo. Molecular Cancer Re-

Journal

of

Cancer

2007;

120:

1418–1425.

66. Yamada T, Takaoka AS, Naishiro Y, et al.

search 2012; 10: 250–258.

54. Hurlin P, Foley K, Ayer D, Eisenman R,

Transactivation of the multidrug resis-

76. Hatsell S, Rowlands T, Hiremath M,

Hanahan D, Arbeit J. Regulation of Myc

tance 1 gene by T-cell factor 4/β-catenin

Cowin P. β-catenin and Tcfs in mammary




45

development and cancer. Journal of Mam-

osteolytic bone metastasis of breast cancer

98. Lefort K, Mandinova A, Ostano P, et al.

mary Gland Biology and Neoplasia 2003; 8:

by engaging notch signaling in bone cells.

Notch1 is a p53 target gene involved in hu-

145–158.

Cancer Cell 2011; 19: 192–205. DOI:

man

10.1016/j.ccr.2010.12.022.

through negative regulation of ROCK1/2

77. Fortini ME. Notch signaling: the core pathway and its posttranslational regulation. Developmental Cell 2009; 16: 633–647.

89. Zagouras P, Stifani S, Blaumueller CM, Carcangiu ML, Artavanis-Tsakonas S. Al-

keratinocyte

tumor

suppression

and MRCKα kinases. Genes & Development 2007; 21: 562–577.

78. Artavanis-Tsakonas S, Rand MD, Lake RJ.

terations in Notch signaling in neoplastic

99. Agrawal N, Frederick MJ, Pickering CR,

Notch signaling: cell fate control and sig-

lesions of the human cervix. Proceedings of

et al. Exome sequencing of head and neck

nal integration in development. Science

the National Academy of Sciences 1995; 92:

squamous

1999; 284: 770–776.

6414–6418.

inactivating mutations in NOTCH1. Sci-

cell

carcinoma

reveals

ence 2011; 333: 1154–1157.

79. Aster JC. In brief: Notch signalling in

90. Daniel B, Rangarajan A, Mukherjee G,

health and disease. The Journal of Pathology

Vallikad E, Krishna S. The link between in-

100. Stransky N, Egloff AM, Tward AD, et al.

2014; 232: 1–3.

tegration and expression of human papil-

The mutational landscape of head and

80. Dai Y, Wilson G, Huang B, et al. Silencing

lomavirus type 16 genomes and cellular

neck squamous cell carcinoma. Science

of Jagged1 inhibits cell growth and inva-

changes in the evolution of cervical

sion in colorectal cancer. Cell Death & Dis-

intraepithelial neoplastic lesions. Journal

2011; 333: 1157–1160. 101. Sun W, Gaykalova DA, Ochs MF, et al. Ac-

of General Virology 1997; 78: 1095–1101.

tivation of the NOTCH pathway in head

81. Radtke F, MacDonald HR, Tacchini-Cottier

91. Veeraraghavalu K, Pett M, Kumar RV, et al.

and neck cancer. Cancer Research 2014; 74:

F. Regulation of innate and adaptive im-

Papillomavirus-mediated neoplastic pro-

munity by Notch. Nature Reviews Immunol-

gression is associated with reciprocal

102. Bernardes de Jesus B, Blasco MA. Telome-

ogy 2013; 13: 427–437.

changes in JAGGED1 and manic fringe ex-

rase at the intersection of cancer and ag-

ease 2014; 5: e1170.

82. Hori K, Sen A, Artavanis-Tsakonas S. Notch signaling at a glance. Journal of Cell Science 2013; 126: 2135–2140.

pression linked to notch activation. Journal of Virology 2004; 78: 8687–8700. 92. Vliet-Gregg

PA,

Hamilton

1091–1104.

ing. Trends in Genetics 2013; 29: 513–520. 103. Günes C, Rudolph KL. The role of telo-

JR,

meres in stem cells and cancer. Cell 2013;

83. Capaccione KM, Pine SR. The Notch sig-

Katzenellenbogen RA. NFX1-123 and hu-

naling pathway as a mediator of tumor

man papillomavirus 16E6 increase notch

104. Mocellin S, Pooley KA, Nitti D. Telome-

expression in keratinocytes. Journal of Vi-

rase and the search for the end of cancer.

rology 2013; 87: 13741–13750.

Trends in Molecular Medicine 2013; 19:

Carcinogenesis

survival.

2013;

34:

1420–1430.

152: 390–393.

84. Santagata S, Demichelis F, Riva A, et al.

93. Weijzen S, Zlobin A, Braid M, Miele L,

JAGGED1 expression is associated with

Kast WM. HPV16 E6 and E7 oncoproteins

105. Xu L, Li S, Stohr BA. The role of telomere

prostate cancer metastasis and recurrence.

regulate Notch-1 expression and cooperate

biology in cancer. Annual Review of Pathol-

Cancer Research 2004; 64: 6854–6857. DOI:

to induce transformation. Journal of Cellu-

10.1158/0008-5472.CAN-04-2500.

lar Physiology 2003; 194: 356–362.

125–133.

ogy: Mechanisms of Disease 2013; 8: 49–78. 106. Bodnar AG, Ouellette M, Frolkis M, et al.

85. Purow BW, Haque RM, Noel MW, et al.

94. Talora C, Sgroi DC, Crum CP, Dotto GP.

Extension of life-span by introduction of

Expression of Notch-1 and its ligands,

Specific down-modulation of Notch1 sig-

telomerase into normal human cells. Sci-

Delta-like-1 and Jagged-1, is critical for gli-

naling in cervical cancer cells is required

oma cell survival and proliferation. Cancer

for sustained HPV-E6/E7 expression

107. Hahn WC, Stewart SA, Brooks MW, et al.

Research

and late steps of malignant transforma-

Inhibition of telomerase limits the growth

tion. Genes & Development 2002; 16:

of human cancer cells. Nature Medicine

2005;

65:

2353–2363.

DOI:

10.1158/0008-5472.CAN-04-1890. 86. Reedijk M, Odorcic S, Chang L, et al. High-

2252–2263.

ence 1998; 279: 349–352.

1999; 5: 1164–1170.

level coexpression of JAG1 and NOTCH1

95. Wang NJ, Sanborn Z, Arnett KL, et al.

108. Klingelhutz AJ, Foster SA, McDougall JK.

is observed in human breast cancer and

Loss-of-function mutations in Notch re-

Telomerase activation by the E6 gene

is associated with poor overall survival.

ceptors in cutaneous and lung squamous

product of human papillomavirus type

Cancer Research 2005; 65: 8530–8537. DOI:

cell carcinoma. Proceedings of the National

10.1158/0008-5472.CAN-05-1069.

Academy

87. Lin JT, Chen MK, Yeh KT, et al. Association

of

Sciences

2011;

108:

17761–17766.

of high levels of Jagged-1 and Notch-1 ex-

96. Klinakis A, Lobry C, Abdel-Wahab O, et al.

pression with poor prognosis in head and

A novel tumour-suppressor function for

neck cancer. Annals of Surgical Oncology

the Notch pathway in myeloid leukaemia.

2010;

Nature 2011; 473: 230–233.

17:

2976–2983.

DOI:

10.1245/

s10434-010-1118-9. 88. Sethi N, Dai X, Winter CG, Kang Y. Tumor-derived

JAGGED1

promotes

16. 1996. 109. Veldman T, Horikawa I, Barrett JC, Schlegel R. Transcriptional activation of the telomerase

hTERT

gene

by

human

papillomavirus type 16 E6 oncoprotein. Journal of Virology 2001; 75: 4467–4472. 110. Liu X, Dakic A, Zhang Y, Dai Y, Chen R,

97. Network CGAR. Comprehensive genomic

Schlegel R. HPV E6 protein interacts phys-

characterization of squamous cell lung

ically and functionally with the cellular tel-

cancers. Nature 2012; 489: 519-525.

omerase


complex.

Proceedings

of

the


46

J. Chen National Academy of Sciences 2009; 106:

target genes in leukemia. Molecular Medi-

Sequence evolution of the intrinsically dis-

18780–18785.

cine Reports 2014; 9: 1283–1288.

ordered and globular domains of a model

111. Ling H, Fabbri M, Calin GA. MicroRNAs

122. Kastl L, Brown I, Schofield A. miRNA-

and other non-coding RNAs as targets for

34a is associated with docetaxel resis-

anticancer drug development. Nature Re-

tance in human breast cancer cells. Breast

views Drug Discovery 2013; 12: 847–865.

Cancer Research and Treatment 2012; 131:

112. Martinez I, Gardiner A, Board K, Monzon

viral oncoprotein. PloS One 2012; 7: e47661. 133. Roman A, Munger K. The papillomavirus E7 proteins. Virology 2013; 445: 138–168. 134. Dick FA, Rubin SM. Molecular mecha-

445–454.

F, Edwards R, Khan S. Human papilloma-

123. Du R, Sun W, Xia L, et al. Hypoxia-induced

nisms underlying RB protein function. Na-

virus type 16 reduces the expression of

down-regulation of microRNA-34a pro-

ture Reviews Molecular Cell Biology 2013; 14:

microRNA-218 in cervical carcinoma cells.

motes EMT by targeting the Notch signal-

Oncogene 2008; 27: 2575–2582.

ing pathway in tubular epithelial cells.

113. Yamamoto N, Kinoshita T, Nohata N, et al.

297–306. 135. Rubin SM. Deciphering the retinoblastoma protein phosphorylation code. Trends in

PloS One 2012; 7: e30771.

Biochemical Sciences 2013; 38: 12–19.

Tumor suppressive microRNA-218 in-

124. Ribeiro J, Sousa H. MicroRNAs as bio-

hibits cancer cell migration and invasion

markers of cervical cancer development: a

136. Di Fiore R, D’Anneo A, Tesoriere G, Vento

by targeting focal adhesion pathways in

literature review on miR-125b and miR-34a.

R. RB1 in cancer: different mechanisms of

cervical squamous cell carcinoma. Interna-

Molecular Biology Reports 2014; 41: 1525–1531.

RB1 inactivation and alterations of pRb

tional Journal of Oncology 2013; 42: 1523–

125. Cui F, Li X, Zhu X, et al. MiR-125b inhibits

pathway in tumorigenesis. Journal of Cellu-

tumor growth and promotes apoptosis of

1532. 114. Wang X, Wang H-K, McCoy JP, et al. Onco-

lar Physiology 2013; 228: 1676–1687.

targeting

137. Park JW, Shin M-K, Pitot HC, Lambert PF.

genic HPV infection interrupts the expres-

phosphoinositide 3-kinase catalytic sub-

High incidence of HPV-associated head

sion

unit delta. Cellular Physiology and Biochem-

and neck cancers in FA deficient mice is as-

istry 2012; 30: 1310–1318.

sociated with E7’s induction of DNA dam-

of

tumor-suppressive

miR-34a

through viral oncoprotein E6. RNA 2009; 15: 637–647.

cervical

cancer

cells

by

126. Nuovo GJ, Wu X, Volinia S, et al. Strong in-

age through its inactivation of pocket proteins. PloS One 2013; 8: e75056.

115. Zheng Z-M, Wang X. Regulation of cellu-

verse correlation between microRNA-125b

lar miRNA expression by human papillo-

and human papillomavirus DNA in pro-

138. Münger K, Werness B, Dyson N, Phelps W,

maviruses. Biochimica et Biophysica Acta

ductive infection. Diagnostic Molecular Pa-

Harlow E, Howley P. Complex formation of

(BBA)-Gene Regulatory Mechanisms 2011;

thology 2010; 19: 135–143.

human papillomavirus E7 proteins with the

1809: 668–677.

127. Wang Y, Cai N, Wu X, Cao H, Xie L,

retinoblastoma tumor suppressor gene product. The EMBO Journal 1989; 8: 4099–4105.

116. Chen F, Hu SJ. Effect of microRNA-34a in

Zheng P. OCT4 promotes tumorigenesis

cell cycle, differentiation, and apoptosis: a

and inhibits apoptosis of cervical cancer

139. Ghittoni R, Accardi R, Hasan U, Gheit T,

review. Journal of Biochemical and Molecular

cells by miR-125b/BAK1 pathway. Cell

Sylla B, Tommasino M. The biological

Toxicology 2012; 26: 79–86.

Death & Disease 2013; 4: e760.

properties of E6 and E7 oncoproteins from

117. Hermeking H. The miR-34 family in can-

128. Banzhaf-Strathmann J, Edbauer D. Good

cer and apoptosis. Cell Death & Differentia-

guy or bad guy: the opposing roles of

tion 2010; 17: 193–199. 118. Li B, Hu Y, Ye F, Li Y, Lv W, Xie X. Re-

microRNA 125b in cancer. Cell Communication and Signaling 2014; 12: 30.

human papillomaviruses. Virus Genes 2010; 40: 1–13. 140. Münger K, Basile JR, Duensing S, et al. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein.

duced miR-34a expression in normal cervi-

129. Jung HM, Phillips BL, Chan EK. miR-375

cal tissues and cervical lesions with high-

activates p21 and suppresses telomerase

risk human papillomavirus infection. In-

activity by coordinately regulating HPV

141. McLaughlin-Drubin ME, Münger K. The

ternational Journal of Gynecological Cancer

E6/E7, E6AP, CIP2A, and 14-3-3zeta. Mo-

human papillomavirus E7 oncoprotein. Vi-

2010; 20: 597–604.

lecular Cancer 2014; 13: 80.

Oncogene 2001; 20: 7888–7898.

rology 2009; 384: 335–344.

119. Xie X, Piao L, Bullock B, et al. Targeting

130. Tsukamoto Y, Nakada C, Noguchi T, et al.

HPV16 E6-p300 interaction reactivates

MicroRNA-375 is downregulated in gas-

man

p53 and inhibits the tumorigenicity of

tric carcinomas and regulates cell survival

oncoprotein associates with the cullin 2

HPV-positive head and neck squamous cell

by targeting PDK1 and 14-3-3ζ. Cancer Re-

ubiquitin ligase complex, which contrib-

carcinoma. Oncogene 2013; 33: 1037–1046.

search 2010; 70: 2339–2349.

utes to degradation of the retinoblastoma

120. Wang X, Meyers C, Guo M, Zheng ZM.

131. Wang

X,

Wang

H-K,

Li

Y,et

al.

142. Huh K, Zhou X, Hayakawa H, et al. Hupapillomavirus

type

16

E7

tumor suppressor. Journal of Virology 2007;

Upregulation of p18Ink4c expression by

microRNAs are biomarkers of oncogenic

oncogenic HPV E6 via p53-miR-34a path-

human papillomavirus infections. Proceed-

143. Heck DV, Yee CL, Howley PM, Münger K.

way. International Journal of Cancer 2011;

ings of the National Academy of Sciences

Efficiency of binding the retinoblastoma

129: 1362–1372.

2014; 111: 4262–4267.

protein correlates with the transforming

81: 9737–9747.

121. Tang R, Li J, Yue M, et al. A correlation

132. Chemes LB, Glavina J, Alonso LG, Ma-

capacity of the E7 oncoproteins of the hu-

analysis of miRNA 34a and its predicted

rino-Buslje C, de Prat-Gay G, Sánchez IE.

man papillomaviruses. Proceedings of the



Signaling pathways in HPV-associated cancers National Academy of Sciences 1992; 89: 4442–4446. 144. Sang B-C, Barbosa MS. Single amino acid

47 Rho

7d and miR-205 are prognostic markers of

GTPases in cell biology. Nature 2002; 420:

head and neck squamous cell carcinoma.

629–635.

The American Journal of Pathology 2009;

154. Etienne-Manneville

S,

Hall

A.

174: 736–745.

substitutions in" low-risk" human papillo-

155. Ridley AJ. Rho GTPases and actin dynam-

mavirus (HPV) type 6 E7 protein enhance

ics in membrane protrusions and vesicle

166. Xie H, Zhao Y, Caramuta S, Larsson C,

features characteristic of the" high-risk"

trafficking. Trends in Cell Biology 2006; 16:

Lui W-O. miR-205 expression promotes

HPV E7 oncoproteins. Proceedings of the

522–529.

cell proliferation and migration of human

National Academy of Sciences 1992; 89:

156. Wojnacki J, Quassollo G, Marzolo MP, Cáceres A. Rho GTPases at the crossroad

8063–8067.

cervical cancer cells. PloS One 2012; 7: e46990. 167. Tong X, Xie D, O’Kelly J, Miller CW,

145. Hwang SG, Lee D, Kim J, Seo T, Choe J.

of signaling networks in mammals: Im-

Human papillomavirus type 16 E7 binds

pact of Rho-GTPases on microtubule or-

Muller-Tidow C, Koeffler HP. Cyr61, a

to E2F1 and activates E2F1-driven tran-

ganization and dynamics. Small GTPases

member of CCN family, is a tumor sup-

2014; 5: 0-1.

pressor in non-small cell lung cancer.

scription in a retinoblastoma proteinindependent manner. Journal of Biological

157. Todorovic B, Nichols AC, Chitilian JM, et al. The human papillomavirus E7 pro-

Chemistry 2002; 277: 2923–2930.

Journal of Biological Chemistry 2001; 276: 47709–47714.

alter

168. Chuang J-Y, Yang W-Y, Lai C-H, Lin C-D,

Münger K. Human papillomavirus type

p190RhoGAP function. Journal of Virology

Tsai M-H, Tang C-H. CTGF inhibits cell

16 E7 oncoprotein associates with E2F6.

2014; JVI: 03263–03213.

motility and COX-2 expression in oral can-

146. McLaughlin-Drubin

ME,

Huh

K-W,

Journal of Virology 2008; 82: 8695–8705. 147. Collins AS, Nakahara T, Do A, Lambert

teins

associate

with

and

158. Melar-New M, Laimins LA. Human papillomaviruses

modulate

expression

of

cer cells. International Immunopharmacology 2011; 11: 948–954.

PF. Interactions with pocket proteins

microRNA 203 upon epithelial differentia-

169. Ofir M, Hacohen D, Ginsberg D. MiR-15

contribute to the role of human papillo-

tion to control levels of p63 proteins. Jour-

and miR-16 are direct transcriptional tar-

mavirus type 16 E7 in the papillomavi-

nal of Virology 2010; 84: 5212–5221.

gets of E2F1 that limit E2F-induced prolif-

rus life cycle. Journal of Virology 2005;

159. Bo J, Yang G, Huo K,et al. microRNA-203 suppresses bladder cancer development

79: 14769–14780. 148. Shin M-K, Sage J, Lambert PF. Inactivating all three Rb family pocket proteins is insufficient to initiate cervical cancer. Cancer Research 2012; 72: 5418–5427. 149. Menges CW, Baglia LA, Lapoint R, McCance DJ. Human papillomavirus type 16 E7 up-regulates AKT activity through the retinoblastoma protein. Cancer Research 2006; 66: 5555–5559.

by repressing bcl-w expression. FEBS Journal 2011; 278: 786–792.

eration by targeting cyclin E. Molecular Cancer Research 2011; 9: 440-447. 170. Myklebust M, Bruland O, Fluge Ø, Skarstein A, Balteskard L, Dahl O.

160. Bian K, Fan J, Zhang X, et al. MicroRNA-

MicroRNA-15b is induced with E2F-

203 leads to G1 phase cell cycle arrest in la-

controlled genes in HPV-related cancer.

ryngeal

British

carcinoma

cells

by

directly

targeting survivin. FEBS Letters 2012; 586:

Journal

of

Cancer

2011;

105:

1719–1725. 171. Wang X, Tang S, Le S-Y, et al. Aberrant ex-

804–809. 161. Takeshita N, Mori M, Kano M, et al. miR-

pression

of

oncogenic

and

tumor-

203 inhibits the migration and invasion of

suppressive microRNAs in cervical cancer

150. Pim D, Massimi P, Dilworth SM, Banks L.

esophageal squamous cell carcinoma by

is required for cancer cell growth. PloS One

Activation of the protein kinase B pathway

regulating LASP1. International Journal of

by the HPV-16 E7 oncoprotein occurs

Oncology 2012; 41: 1653–1661.

2008; 3: e2557. 172. Aqeilan R, Calin G, Croce C. miR-15a and

through a mechanism involving interac-

162. McKenna DJ, Patel D, McCance DJ. miR-

miR-16-1 in cancer: discovery, function

tion with PP2A. Oncogene 2005; 24:

24 and miR-205 expression is dependent

and future perspectives. Cell Death & Dif-

7830–7838.

on

HPV onco-protein

expression

in

ferentiation 2010; 17: 215–220.

151. Liu J, Wang X, Zhou G, et al. Cancerous in-

keratinocytes. Virology 2014; 448: 210–216.

hibitor of protein phosphatase 2A is

163. Wang Z, Liao H, Deng Z, et al. miRNA-205

The miR-15a–miR-16-1 cluster controls

overexpressed in cervical cancer and up-

affects infiltration and metastasis of breast

prostate cancer by targeting multiple on-

regulated by human papillomavirus 16

cancer. Biochemical and Biophysical Research

cogenic activities. Nature Medicine 2008;

E7

oncoprotein.

Gynecologic

Oncology

2011; 122: 430–436.

Communications 2013; 441: 139–143.

173. Bonci D, Coppola V, Musumeci M, et al.

14: 1271–1277.

164. Dar AA, Majid S, de Semir D, Nosrati M,

174. Wang F, Fu X-D, Zhou Y, Zhang Y. Down-

152. Charette S, McCance D. The E7 protein

Bezrookove V, Kashani-Sabet M. miRNA-

regulation of the cyclin E1 oncogene ex-

from human papillomavirus type 16 en-

205 suppresses melanoma cell prolifera-

pression by microRNA-16-1 induces cell

hances keratinocyte migration in an Akt-

tion and induces senescence via regulation

cycle arrest in human cancer cells. BMB

dependent manner. Oncogene 2007; 26:

of E2F1 protein. Journal of Biological Chem-

7386–7390.

istry 2011; 286: 16606–16614.

153. Hall A. Rho GTPases and the actin cytoskeleton. Science 1998; 279: 509–514.

Reports 2009; 42: 725–730. 175. Krawczyk E, Suprynowicz FA, Liu X, et al.

165. Childs G, Fazzari M, Kung G, et al. Low-

Koilocytosis: a cooperative interaction be-

let-

tween the human papillomavirus E5 and

level


expression

of

MicroRNAs


48

J. Chen E6 oncoproteins. The American Journal of Pathology 2008; 173: 682–688.

176. Jost M, Kari C, Rodeck U. The EGF receptor - an essential regulator of multiple epidermal functions. European Journal of Dermatology 2000; 10: 505–510.

185. Villa-Morales M, Fernández-Piqueras J.

196. Zubillaga-Guerrero M, Illades-Aguiar B,

Targeting the Fas/FasL signaling pathway

Leyva-Vazquez M, et al. The integration

in cancer therapy. Expert Opinion on Thera-

of HR-HPV increases the expression of

peutic Targets 2012; 16: 85–101.

cyclins A and E in cytologies with and

186. Kabsch K, Alonso A. The human papillo-

without low-grade lesions. Journal of

mavirus type 16 E5 protein impairs

Cytology/Indian

177. Jost M, Huggett TM, Kari C, Boise LH,

TRAIL-and FasL-mediated apoptosis in

2013; 30: 1–7.

Rodeck U. Epidermal growth factor

HaCaT cells by different mechanisms. Jour-

receptor-dependent control of keratinocyte

nal of Virology 2002; 76: 12162–12172.

of the HPV16 E7 oncoprotein with cyclin

survival and Bcl-xL expression through a

187. Kabsch K, Mossadegh N, Kohl A, et al. The

A/CDK2 and cyclin E/CDK2 complexes.

MEK-dependent pathway. The Journal of

HPV-16 E5 protein inhibits TRAIL-and

Biological Chemistry 2001; 276: 6320–6326.-

FasL-mediated

human

198. Yoshinouchi M, Yamada T, Kizaki M, et al.

DOI:

keratinocyte raft cultures. Intervirology

In vitro and in vivo growth suppression of

2004; 47: 48–56.

human papillomavirus 16-positive cervi-

10.1074/jbc.M008210200

DOI:

10.1074/jbc.M008210200.

apoptosis

in

178. Chen J, Elfiky A, Han M, Chen C, Saif

188. Regan JA, Laimins LA. Bap31 is a novel

MW. The role of Src in colon cancer and

target of the human papillomavirus E5

its therapeutic implications. Clinical Colo-

protein. Journal of Virology 2008; 82:

rectal Cancer 2014; 13: 5–13.

of

Cytologists

197. Nguyen CL, Münger K. Direct association

Virology 2008; 380: 21–25.

cal cancer cells by E6 siRNA. Molecular Therapy 2003; 8: 762–768. 199. Jonson AL, Rogers LM, Ramakrishnan S, Downs Jr LS. Gene silencing with siRNA

10042–10051.

179. Pim D, Collins M, Banks L. Human papil-

Academy

189. Iwasawa R, Mahul-Mellier AL, Datler C,

targeting E6/E7 as a therapeutic interven-

lomavirus type 16 E5 gene stimulates the

Pazarentzos

transforming activity of the epidermal

Bap31 bridge the mitochondria–ER inter-

growth factor receptor. Oncogene 1992; 7:

face to establish a platform for apoptosis

200. Debnath J, Walker SJ, Brugge JS. Akt acti-

27–32.

induction. The EMBO Journal 2011; 30:

vation disrupts mammary acinar architec-

556–568.

ture and enhances proliferation in an

180. Crusius K, Auvinen E, Steuer B, Gaissert

E,

Grimm

S.

Fis1

and

tion in a mouse model of cervical cancer. Gynecologic Oncology 2008; 111: 356–364.

mTOR-dependent manner. The Journal of

H, Alonso A. The human papillomavirus

190. Kotnik Halavaty K, Regan J, Mehta K,

type 16 E5-protein modulates ligand-

Laimins L. Human papillomavirus E5

dependent activation of the EGF receptor

oncoproteins bind the A4 endoplasmic re-

201. Coppock JD, Wieking BG, Molinolo AA,

family in the human epithelial cell line

ticulum protein to regulate proliferative

Gutkind JS, Miskimins WK, Lee JH. Im-

HaCaT. Experimental Cell Research 1998;

ability upon differentiation. Virology 2014;

proved clearance during treatment of

241: 76–83.

452: 223–230.

HPV-positive

181. Martin P, Vass WC, Schiller JT, Lowy DR,

191. Hanahan D, Weinberg RA. Hallmarks of

Velu TJ. The bovine papillomavirus E5

cancer: the next generation. Cell 2011; 144:

transforming protein can stimulate the

646–674.

transforming activity of EGF and CSF-1 receptors. Cell 1989; 59: 21–32. 182. Kim S-H, Juhnn Y-S, Kang S, et al. Hu-

Cell Biology 2003; 163: 315–326.

head

and

neck

cancer

through mTOR inhibition. Neoplasia (New York, NY) 2013; 15: 620–630. 202. Conesa-Zamora P, Doménech-Peris A,

192. Bretones G, Delgado MD, León J. Myc and

Orantes-Casado FJ, et al. Effect of human

cell cycle control. Biiochimica et Biophysica Acta

papillomavirus on cell cycle–related pro-

(BBA) - Gene Regulatory Mechanisms 2014.

teins p16, Ki-67, Cyclin D1, p53, and

man papillomavirus 16 E5 up-regulates

DOI: 10.1016/j.pharmthera.2013.12.004.

ProEx C in precursor lesions of cervical

the expression of vascular endothelial

193. Cho NH, Kang S, Hong S, et al. Elevation

carcinoma A tissue microarray study.

growth factor through the activation of

of cyclin B1, active cdc2, and HuR in cervi-

American Journal of Clinical Pathology

epidermal

receptor,

cal neoplasia with human papillomavirus

MEK/ERK1, 2 and PI3K/Akt. Cellular and

type 18 infection. Cancer Letters 2006; 232:

growth

factor

Molecular Life Sciences 2006; 63: 930–938.

170–178.

2009; 132: 378–390. 203. Portari EA, Russomano FB, de Camargo MJ, et al. Immunohistochemical expression

183. Crusius K, Rodriguez I, Alonso A. The hu-

194. Hashiguchi Y, Tsuda H, Nishimura S,

of cyclin D1, p16Ink4a, p21WAF1, and Ki-

man papillomavirus type 16 E5 protein

Inoue T, Kawamura N, Yamamoto K. Re-

67 correlates with the severity of cervical

modulates ERK1/2 and p38 MAP kinase

lationship between HPV typing and the

neoplasia. International Journal of Gyneco-

activation by an EGFR-independent pro-

status of G2 cell cycle regulators in cervi-

cess in stressed human keratinocytes. Vi-

cal neoplasia. Oncology Reports 2004; 12:

rus Genes 2000; 20: 65–69. 184. Yuan

C-H,

Filippova

587–591. M,

logic Pathology 2013; 32: 501–508. 204. Butz K, Denk C, Ullmann A, Scheffner M, Hoppe-Seyler F. Induction of apoptosis in

Duerksen-

195. Cho NH, Lim SY, Kim YT, Kim D, Kim YS,

human papillomavirus positive cancer

Hughes P. Modulation of apoptotic path-

Kim JW. G2 checkpoint in uterine cervical

cells by peptide aptamers targeting the

ways by human papillomaviruses (HPV):

cancer with HPV 16 E6 according to p53

viral E6 oncoprotein. Proceedings of the

mechanisms and implications for therapy.

polymorphism and its screening value.

National Academy of Sciences 2000; 97:

Viruses 2012; 4: 3831–3850.

Gynecologic Oncology 2003; 90: 15–22.

6693–6697.



Signaling pathways in HPV-associated cancers 205. Qi Z, Xu X, Zhang B, et al. Effect of simultaneous silencing of HPV-18 E6 and E7 on in-

49 Polo-like kinase 4 expression. Molecular Cancer 2011; 10: 61.

226. Gottesman MM. Mechanisms of cancer drug resistance. Annual Review of Medicine 2002; 53: 615–627.

ducing apoptosis in HeLa cells this paper is

215. Habedanck R, Stierhof Y-D, Wilkinson CJ,

one of a selection of papers published in

Nigg EA. The Polo kinase Plk4 functions

227. Longley D, Johnston P. Molecular mecha-

this special issue entitled “Second Interna-

in centriole duplication. Nature Cell Biology

nisms of drug resistance. The Journal of Pa-

tional Symposium on Recent Advances in

2005; 7: 1140–1146.

Basic, Clinical, and Social Medicine” and

thology 2005; 205: 275–292.

216. Yu JJ, Fu P, Pink JJ, et al. HPV infection

228. Luqmani Y. Mechanisms of drug resistance in cancer chemotherapy. Medical

has undergone the Journal’s usual peer re-

and

view process. Biochemistry and Cell Biology

HIV-infected East African patients with

Principles and Practice 2008; 14: 35–48.

2010; 88: 697–704.

conjunctival carcinoma. PloS One 2010;

229. Chen J, Huang X-F, Qiao L, Katsifis A. In-

5: e10477. DOI: 10.1371/journal.pone.00

sulin caused drug resistance to oxaliplatin

10477.

in colon cancer cell line HT29. Journal of

206. Smeets SJ, van der Plas M, Schaaij-Visser T, et al. Immortalization of oral keratinocytes

EGFR

activation/alteration

in

Gastrointestinal Oncology 2011; 2: 27–33.

by functional inactivation of the p53 and

217. Kersemaekers A-MF, Fleuren GJ, Kenter

pRb pathways. International Journal of Can-

GG, et al. Oncogene alterations in carcino-

230. Chen J, Katsifis A, Hu C, Huang X-F. Insu-

cer 2011; 128: 1596–1605.

mas of the uterine cervix: overexpression

lin decreases therapeutic efficacy in colon

207. Thomas M, Banks L. Inhibition of Bak-

of the epidermal growth factor receptor is

cancer cell line HT29 via the activation of

induced apoptosis by HPV-18 E6. Onco-

associated with poor prognosis. Clinical

the PI3K/Akt pathway. Current Drug Dis-

gene 1998; 17: 2943–2954.

Cancer Research 1999; 5: 577–586.

covery Technologies 2011; 8: 119–125.

208. Guirouilh-Barbat J, Wilhelm T, Lopez BS.

218. Wright AA, Howitt BE, Myers AP, et al.

231. Tsuruo T, Naito M, Tomida A, et al. Molec-

AKT1/BRCA1 in the control of homolo-

Oncogenic mutations in cervical cancer.

ular targeting therapy of cancer: drug re-

gous recombination and genetic stability:

Cancer 2013; 119: 3776–3783.

sistance, apoptosis and survival signal. Cancer Science 2003; 94: 15–21.

the missing link between hereditary and

219. Wierzbicka M, Jozefiak A, Szydlowski J,

sporadic breast cancers. Oncotarget 2010;

et al. Recommendations for the diagnosis

232. Smalley KS, Haass NK, Brafford PA, Lioni

1: 691.

of human papilloma virus (HPV) high

M, Flaherty KT, Herlyn M. Multiple sig-

209. Hernando E, Nahlé Z, Juan G, et al. Rb in-

and low risk in the prevention and treat-

naling pathways must be targeted to over-

activation promotes genomic instability by

ment of diseases of the oral cavity, phar-

come drug resistance in cell lines derived

uncoupling cell cycle progression from mi-

ynx and larynx. Guide of experts PTORL

from melanoma metastases. Molecular

totic control. Nature 2004; 430: 797–802.

and KIDL. Otolaryngologia Polska. The Pol-

Cancer Therapeutics 2006; 5: 1136–1144.

ish Otolaryngology 2013; 67: 113–134. DOI:

233. Xia S, Zhao Y, Yu S, Zhang M. Activated

210. Tsou M-FB, Wang W-J, George KA, Uryu K, Stearns T, Jallepalli PV. Polo kinase

10.1016/j.otpol.2013.01.003.

PI3K/Akt/COX-2 pathway induces resis-

and separase regulate the mitotic licensing

220. Anderson JA, Irish JC, McLachlin CM,

tance to radiation in human cervical can-

of centriole duplication in human cells. De-

Ngan BY. H-ras oncogene mutation and

cer HeLa cells. Cancer Biotherapy &

velopmental Cell 2009; 17: 344–354.

human papillomavirus infection in oral

211. Duensing S, Duensing A, Crum CP, Münger K. Human papillomavirus type

carcinomas. Archives of Otolaryngology-Head & Neck Surgery 1994; 120: 755–760.

Radiopharmaceuticals 2010; 25: 317–323. 234. Wu H-H, Wu J-Y, Cheng Y-W, et al. cIAP2 upregulated by E6 Oncoprotein

16 E7 oncoprotein-induced abnormal cen-

221. Ojesina AI, Lichtenstein L, Freeman SS, et al.

trosome synthesis is an early event in the

Landscape of genomic alterations in cervical

receptor/phosphatidylinositol

evolving malignant phenotype. Cancer Re-

via

epidermal

growth

factor 3-kinase/

carcinomas. Nature 2014; 506: 371–375.

AKT pathway confers resistance to cis-

222. Holohan C, Van Schaeybroeck S, Longley

platin in human papillomavirus 16/18–

212. Duensing A, Chin A, Wang L, Kuan S-F,

DB, Johnston PG. Cancer drug resistance:

infected lung cancer. Clinical Cancer Re-

Duensing S. Analysis of centrosome

an evolving paradigm. Nature Reviews

overduplication in correlation to cell divi-

Cancer 2013; 13: 714–726.

search 2001; 61: 2356–2360.

search 2010; 16: 5200–5210. 235. Lin G, Chen Q, Yu S, et al. Overexpression

sion errors in high-risk human papilloma-

223. Chen J, Shao R, Zhang XD, Chen C. Appli-

of human telomerase reverse transcriptase

virus (HPV)-associated anal neoplasms.

cations of nanotechnology for melanoma

C-terminal polypeptide sensitizes HeLa

Virology 2008; 372: 157–164.

treatment, diagnosis, and theranostics. In-

cells to 5-fluorouracil induced growth in-

ternational Journal of Nanomedicine 2013; 8:

hibition and apoptosis. Molecular Medicine

213. Korzeniewski N, Zheng L, Cuevas R, et al. Cullin 1 functions as a centrosomal sup-

2677.

Reports 2014; 9: 279–284.

pressor of centriole multiplication by regu-

224. Dean M, Fojo T, Bates S. Tumour stem cells

236. Chen J. Targeted therapy of obesity-

lating polo-like kinase 4 protein levels.

and drug resistance. Nature Reviews Cancer

associated colon cancer. Translational Gas-

Cancer Research 2009; 69: 6668–6675.

2005; 5: 275–284.

214. Korzeniewski N, Treat B, Duensing S. The

trointestinal Cancer 2011; 1: 44–57.

225. Chen J, Raymond K. Nuclear receptors,

237. Downward J. Targeting RAS signalling

HPV-16 E7 oncoprotein induces centriole

bile-acid detoxification, and cholestasis.

pathways in cancer therapy. Nature Re-

multiplication through deregulation of

The Lancet 2006; 367: 454–456.

views Cancer 2003; 3: 11–22.



50

J. Chen

238. Chen J. Is Src the key to understanding metastasis and developing new treatments

approaches for cervical cancer. Cancer 2009;

for colon cancer? Nature Clinical Practice.

250. Duenãs-González A, Cetina L, Coronel J,

Gastroenterology & Hepatology 2008; 5:

Martínez-Baños D. Pharmacotherapy op-

306–307.

tions for locally advanced and advanced

239. Eijsink JJ, Noordhuis MG, ten Hoor KA, et al. The epidermal growth factor receptor

cervical cancer. Drugs 2010; 70: 403–432. cisplatin

and

5-

inactivating lentivirus vector. Journal of Virology 1998; 72: 8150–8157.

phase

metastasis and survival in early-stage cer-

fluorouracil with allopurinol for recurrent

261. Logan AC, Haas DL, Kafri T, Kohn DB.

vical cancer. Human Pathology 2010; 41:

or metastatic carcinoma of the uterine cer-

Integrated self-inactivating lentiviral vec-

1735–1741.

vix: a Southwest oncology group trial. Gy-

tors produce full-length genomic tran-

necologic Oncology 1990; 37: 354–358.

scripts competent for encapsidation and

lencing of PTPRR activates MAPK signal-

252. Kaern J, Trope C, Abeler V, Iversen T,

integration. Journal of Virology 2004; 78:

ing, promotes metastasis and serves as a

Kjørstad K. A phase II study of 5-

8421–8436. DOI: 10.1128/JVI.78.16.8421–

biomarker of invasive cervical cancer. On-

fluorouracil/cisplatinum in recurrent cer-

8436.2004..

cogene 2012; 32: 15–26.

vical cancer. Acta Oncologica 1990; 29:

241. Amine A, Rivera S, Opolon P, et al. Novel

of

260. Miyoshi H, Blomer U, Takahashi M, Gage

pathway in relation to pelvic lymph node

240. Su P, Lin Y, Huang R, et al. Epigenetic si-

trial

sion. Molecular Therapy 2000; 1: 516–521. DOI: 10.1006/mthe.2000.0083. FH, Verma IM. Development of a self-

251. Weiss GR, Green S, Hannigan EV, et al. A II

259. Kafri T, van Praag H, Gage FH, Verma IM. Lentiviral vectors: regulated gene expres-

115: 3166–3180.

262. Gu W, Putral L, Hengst K, et al. Inhibition

25–28.

of

cervical

cancer

cell

growth

anti-metastatic action of cidofovir medi-

253. Bonomi P, Blessing J, Ball H, Hanjani P,

in vitro and in vivo with lentiviral-vector

ated by inhibition of E6/E7, CXCR4 and

DiSaia PJ. A phase II evaluation of cis-

delivered short hairpin RNA targeting

Rho/ROCK signaling in HPV+ tumor

platin and 5-fluorouracil in patients with

human papillomavirus E6 and E7 onco-

cells. PloS One 2009; 4: e5018.

advanced squamous cell carcinoma of

genes. Cancer Gene Therapy 2006; 13:

242. Reya T, Morrison SJ, Clarke MF, Weissman

the cervix: a gynecologic oncology group

IL. Stem cells, cancer, and cancer stem

study. Gynecologic Oncology 1989; 34:

cells. Nature 2001; 414: 105–111.

1023–1032. DOI: 10.1038/sj.cgt.7700971. 263. Gu W, Payne E, Sun S, Burgess M, McMillan NA. Inhibition of cervical cancer cell

357–359. C,

growth in vitro and in vivo with dual

therapeutic promise of the cancer stem cell

Leelaphatanadit C, Therasakvichya S,

shRNAs. Cancer Gene Therapy 2011; 18:

concept. The Journal of Clinical Investigation

Inthasorn P. A pilot phase II study of cap-

2010; 120: 41.

ecitabine plus cisplatin in the treatment of

264. Tan S, Hougardy BM, Meersma GJ, et al.

recurrent carcinoma of the uterine cervix.

Human papilloma virus 16 e6 RNA inter-

Oncology 2007; 72: 33–38.

ference enhances cisplatin and death

243. Frank NY, Schatton T, Frank MH. The

244. Catalano V, Di Franco S, Iovino F, Dieli F, Stassi G, Todaro M. CD133 as a target for colon cancer. Expert Opinion on Therapeutic Targets 2012; 16: 259–267.

254. Benjapibal

M,

Thirapakawong

255. Errihani H, M’rabti H, Ismaili N, Inrhaoun

219–227. DOI: 10.1038/cgt.2010.72.

receptor-mediated apoptosis in human cervical

245. Tang AL, Owen JH, Hauff SJ, et al. Head

bine and cisplatin in advanced, persistent,

Pharmacology 2012; 81: 701–709. DOI:

and neck cancer stem cells the effect of

or recurrent carcinoma of the cervix. Inter-

HPV—an in vitro and mouse study. Otolar-

national Journal of Gynecological Cancer

yngology–Head and Neck Surgery 2013; 149:

carcinoma

cells.

Molecular

H, Elghissassi I. Phase II trial of capecita-

mol.111.076539. 265. Chen J, McMillan N, Gu W. Intra-tumor injection of lentiviral-vector delivered

2011; 21: 373–377. 256. Ischenko I, Camaj P, Seeliger H, et al. In-

shRNA targeting human papillomavirus

246. Qi W, Zhao C, Zhao L, et al. Sorting and

hibition of Src tyrosine kinase reverts

E6 and E7 oncogenes reduces tumor

identification of side population cells in

chemoresistance toward 5-fluorouracil in

growth in a xenograft cervical cancer

the human cervical cancer cell line HeLa.

human pancreatic carcinoma cells: an in-

model in mice. Journal of Solid Tumors

Cancer Cell International 2014; 14: 3.

volvement of epidermal growth factor re-

252–260.

247. Villanueva-Toledo J, Ponciano-Gómez A, Ortiz-Sánchez E, Garrido E. Side popula-

ceptor

signaling.

Oncogene

2008;

27:

7212–7222.

2012; 2: 4–10. 266. Zhou J, Peng C, Li B, et al. Transcriptional gene silencing of HPV16 E6/E7

tions from cervical-cancer-derived cell

257. Abdulkarim B, Sabri S, Deutsch E, et al.

induces growth inhibition via apoptosis

lines have stem-cell-like properties. Molec-

Antiviral agent cidofovir restores p53

in vitro and in vivo. Gynecologic Oncology

ular Biology Reports 2014; 41: 1993–2004.

function and enhances the radiosensitivity

2012; 124: 296–302. DOI: 10.1016/j.

in HPV-associated cancers. Oncogene 2002;

ygyno.2011.10.028.

248. Zhang M, Kumar B, Piao L, et al. Elevated intrinsic cancer stem cell population in hu-

21: 2334–2346.

267. Faried LS, Faried A, Kanuma T, et al. Pre-

man papillomavirus-associated head and

258. Tan S, de Vries EG, van der Zee AG, de

dictive and prognostic role of activated

neck squamous cell carcinoma. Cancer

Jong S. Anticancer drugs aimed at E6 and

mammalian target of rapamycin in cervi-

2014; 120: 992–1001.

E7 activity in HPV-positive cervical cancer.

cal cancer treated with cisplatin-based

Current Cancer Drug Targets 2012; 12:

neoadjuvant

170–184. DOI: BSP/CCDT/E-Pub/00181.

Reports 2006; 16: 57–64.

249. Movva

S,

Rodriguez

L,

Arias

PH,

Verschraegen C. Novel chemotherapy


chemotherapy.

Oncology



51

268. Chen J. The Src/PI3K/Akt signal path-

squamous cell carcinoma, making the

chemosensitivity in cervical cancer cells

way may play a key role in decreased

EGFR pathway a novel therapeutic

via induction of apoptosis. Life Sciences

drug efficacy in obesity-associated cancer.

target. British Journal of Cancer 2011;

Journal of Cellular Biochemistry 2010; 110:

105: 420–427. 278. Deberne M, Levy A, Mondini M, et al. The

279–280. 269. Cheng H, Walls M, M Baxi S, Yin M-J.

combination

2013; 93: 7–16. 286. Yokoyama M, Noguchi M, Nakao Y, Pater

of

antiviral

gallocatechin gallate effects on growth, ap-

cidofovir

antibody

optosis, and telomerase activity in cervical

malignancy. Current Cancer Drug Targets

cetuximab exerts an antiproliferative effect

cell lines. Gynecologic Oncology 2004; 92:

2013; 13: 267–277.

on HPV-positive cervical cancer cell lines’

tion of Akt as a mechanism for tumor immune evasion. Molecular Therapy 2009; 17:

anti-EGFR

A, Iwasaka T. The tea polyphenol,( )-epi-

agent

Targeting the mTOR pathway in tumor

270. Noh KH, Kang TH, Kim JH, et al. Activa-

and

the

in-vitro and in-vivo xenografts. Anti-Cancer Drugs 2013; 24: 599–608.

197–204. 287. Ahn WS, Huh SW, Bae S-M, et al. A major constituent of green tea, EGCG, in-

279. de Mello RA, Gerós S, Alves MP, Moreira

hibits the growth of a human cervical

F, Avezedo I, Dinis J. Cetuximab plus

cancer cell line, CaSki cells, through apo-

271. Rashmi R, DeSelm C, Helms C, et al. AKT

platinum-based chemotherapy in head

ptosis, G1 arrest, and regulation of gene

inhibitors promote cell death in cervical

and neck squamous cell carcinoma: a ret-

expression. DNA and Cell Biology 2003;

cancer through disruption of mTOR sig-

rospective study in a single comprehen-

naling and glucose uptake. PloS One

sive European Cancer Institution. PloS

439–447.

One 2014; 9: e86697.

2014; 9: e92948.

22: 217–224. 288. Noguchi M, Yokoyama M, Watanabe S, et al. Inhibitory effect of the tea polyphe-

272. Schwarz JK, Payton JE, Rashmi R, et al.

280. Farley J, Sill MW, Birrer M, et al. Phase II

Pathway-specific analysis of gene expres-

study of cisplatin plus cetuximab in ad-

sion data identifies the PI3K/Akt pathway

vanced, recurrent, and previously treated

as a novel therapeutic target in cervical

cancers of the cervix and evaluation of epi-

289. Singh M, Singh R, Bhui K, Tyagi S,

cancer. Clinical Cancer Research 2012; 18:

dermal growth factor receptor immuno-

Mahmood Z, Shukla Y. Tea polyphenols

1464–1471.

histochemical expression: a gynecologic

induce apoptosis through mitochondrial

oncology group study. Gynecologic Oncol-

pathway and by inhibiting nuclear factor-

ogy 2011; 121: 303–308.

κB and Akt activation in human cervical

273. Liu

Y,

Cui

B,

Qiao

Y,

et

al.

Phosphoinositide-3-kinase inhibition enhances radiosensitization of cervical cancer

in

vivo.

International

Journal

of

nol,( )-epigallocatechin

gallate,

on

growth of cervical adenocarcinoma cell lines. Cancer Letters 2006; 234: 135–142.

281. Goncalves A, Fabbro M, Lhomme C, et al.

cancer cells. Oncology Research Featuring

A phase II trial to evaluate gefitinib as

Preclinical and Clinical Cancer Therapeutics

Gynecological Cancer 2011; 21: 100–105.

second-or third-line treatment in patients

274. Tinker A, Ellard S, Welch S, et al. Phase II

with recurring locoregionally advanced

study of temsirolimus (CCI-779) in women

or metastatic cervical cancer. Gynecologic

Gupta

with recurrent, unresectable, locally ad-

Oncology 2008; 108: 42–46.

Curcumin suppresses human papilloma-

M,

Chauhan

SC.

G,

virus oncoproteins, restores p53, rb, and ptpn13 proteins and inhibits benzo [a]

Group (NCIC CTG IND 199). Gynecologic

erlotinib combined with cisplatin and

pyrene-induced upregulation of HPV

Oncology 2013; 130: 269–274.

radiotherapy in patients with locally ad-

E7. Molecular Carcinogenesis 2011; 50:

Improved survival with bevacizumab in advanced cervical cancer. New En-

Moralez

Jaggi

Grazziotin R, et al. Phase 2 trial of

275. Tewari KS, Sill MW, Long III HJ, et al.

A,

BK,

vix. A trial of the NCIC Clinical Trials

vanced or metastatic carcinoma of the cer-

282. Nogueira-Rodrigues

2011; 19: 245–257. 290. Maher DM, Bell MC, O’Donnell EA,

vanced cervical cancer. Cancer 2014; 120: 1187–1193.

47–57. 291. Singh M, Singh N. Curcumin counteracts

283. Callegaro-Filho D, Kavanagh JJ, Nick

the proliferative effect of estradiol and in-

gland Journal of Medicine 2014; 370:

AM,

KM.

duces apoptosis in cervical cancer cells.

734–743.

Sustained complete response after main-

Molecular and Cellular Biochemistry 2011;

Ramirez

PT,

Schmeler

276. Schefter TE, Winter K, Kwon JS, et al. A

tenance therapy with topotecan and

phase II study of bevacizumab in com-

erlotinib for recurrent cervical cancer

292. Roy M, Mukherjee S. Reversal of resis-

bination with definitive radiotherapy

with distant metastases. Case Reports in

tance towards cisplatin by curcumin in

and

Oncology 2014; 7: 97–101.

cervical cancer cells. Asian Pacific Journal

cisplatin

chemotherapy

in

un-

347: 1–11.

of Cancer Prevention: APJCP 2014; 15:

treated patients with locally advanced

284. Nogueira-Rodrigues A, do Carmo CC,

cervical carcinoma: preliminary results

Viegas C, et al. Phase I trial of erlotinib

of RTOG 0417. International Journal of

combined with cisplatin and radiotherapy

Radiation Oncology Biology Physics 2012;

for patients with locally advanced cervical

Molecular

83: 1179–1184.

squamous cell cancer. Clinical Cancer Re-

chemosensitizing efficacy of liposomal

search 2008; 14: 6324–6329.

curcumin in paclitaxel chemotherapy in

277. Iida K, Nakayama K, Rahman M, et al. EGFR gene amplification is related to adverse clinical outcomes in cervical

285. Singh M, Bhui K, Singh R, Shukla Y. Tea


polyphenols

enhance

cisplatin

1403–1410. 293. Sreekanth C, Bava S, Sreekumar E, Anto R. evidences

for

the

mouse models of cervical cancer. Oncogene 2011; 30: 3139–3152.


52

J. Chen

294. Yoysungnoen-Chintana P, Bhattarakosol P,

Patumraj

S.

Antitumor

and

through interferon-γ. Clinical Cancer Research

progression of cervical cancer in a human

2014; 20: 5456–5467.

papillomavirus-transgenic mouse model.

antiangiogenic activities of curcumin in

303. Westermann C, Fischer A, Clad A. Treat-

Proceedings of the National Academy of Sci-

cervical cancer xenografts in nude mice.

ment of vulvar intraepithelial neoplasia

ences of the United States of America 2005;

BioMed Research International 2014: 817972.

with topical 5% imiquimod cream. Interna-

295. Debata PR, Castellanos MR, Fata JE, et al.

tional Journal of Gynecology & Obstetrics

A novel curcumin-based vaginal cream

102: 2490–2495. 313. López-Romero R, Garrido-Guerrero E, Rangel-López A, et al. The cervical malig-

2013; 120: 266–270.

Vacurin selectively eliminates apposed hu-

304. Daayana S, Elkord E, Winters U, et al.

man cervical cancer cells. Gynecologic On-

Phase II trial of imiquimod and HPV ther-

ER-α but retain the ER-β expression. Inter-

cology 2013; 129: 145–153.

apeutic vaccination in patients with vulval

national Journal of Clinical and Experimental

296. Vidya Priyadarsini R, Senthil Murugan R, Maitreyi S, Ramalingam K, Karunagaran D, Nagini S. The flavonoid quercetin in-

intraepithelial neoplasia. British Journal of Cancer 2010; 102: 1129–1136.

Pathology 2013; 6: 1594–1602. 314. Kwasniewska

305. Kenter GG, Welters MJ, Valentijn ARP,

A,

Postawski

K,

Gozdzicka-Jozefiak A, et al. Estrogen

duces cell cycle arrest and mitochondria-

et

HPV-16

and progesterone receptor expression in

mediated apoptosis in human cervical

oncoproteins for vulvar intraepithelial

HPV-positive and HPV-negative cervical

cancer (HeLa) cells through p53 induction

neoplasia. New England Journal of Medicine

carcinomas. Oncology Reports 2011; 26:

and NF-κB inhibition. European Journal of

2009; 361: 1838–1847.

Pharmacology 2010; 649: 84–91.

al.

nant cells display a down regulation of

306. Cheng

297. Bishayee K, Ghosh S, Mukherjee A,

et

al.

Vaccination

W-F,

against

153–160.

Hung

C-F,

C-Y,

315. Chung S-H, Shin MK, Korach KS, Lambert

immunity

and

PF. Requirement for stromal estrogen re-

Sadhukhan R, Mondal J, Khuda-Bukhsh

antiangiogenesis generated by a DNA vac-

ceptor alpha in cervical neoplasia. Hor-

A. Quercetin induces cytochrome-c release

cine encoding calreticulin linked to a tu-

and ROS accumulation to promote apo-

mor

ptosis and arrest the cell cycle in G2/M,

Tumor-specific

Chai

Journal

antigen.

of

Clinical

Investigation 2001; 108: 669–678.

Díaz-Chávez J, et al. Gene expression pro-

in cervical carcinoma: signal cascade and

307. Chuang C-M, Monie A, Hung C-F, Wu T.

drug-DNA interaction. Cell Proliferation

Research treatment with Imiquimod en-

2013; 46: 153–163.

hances antitumor immunity induced by

298. Campo M, Graham S, Cortese M, et al.

mones and Cancer 2013; 4: 50–59. 316. Cortés-Malagón EM, Bonilla-Delgado J,

therapeutic HPV DNA vaccination. 2010.

file

regulated

by

the

HPV16

E7

oncoprotein and estradiol in cervical tissue. Virology 2013; 447: 155–165. 317. Spurgeon ME, Chung S-H, Lambert PF.

HPV-16 E5 down-regulates expression of

308. Peralta-Zaragoza O, Bermúdez-Morales

Recurrence of cervical cancer in mice after

surface HLA class I and reduces recogni-

VH, Pérez-Plasencia C, Salazar-León J,

selective estrogen receptor modulator

tion by CD8 T cells. Virology 2010; 407:

Gómez-Cerón

therapy. The American Journal of Pathology

137–142.

Targeted treatments for cervical cancer:

299. Zhou F, Chen J, Zhao K-N. Human papillomavirus 16-encoded E7 protein inhibits IFN-γ-mediated MHC class I antigen pre-

C,

Madrid-Marina

V.

a review. OncoTargets and Therapy 2012; 5: 315.

2014; 184: 530–540. 318. Munger K. Are selective estrogen receptor modulators (SERMs) a therapeutic option

309. Persson I, Yuen J, Bergkvist L, Schairer C.

for HPV-associated cervical lesions and

by

Cancer incidence and mortality in women

cancers? The American Journal of Pathology

mouse

receiving estrogen and estrogen-progestin

keratinocytes. Journal of General Virology

replacement therapy—long-term follow-

2013; 94: 2504–2514.

up of a Swedish cohort. International Jour-

Hörmann V, Kumi-Diaka J, Rathinavelu

nal of Cancer 1996; 67: 327–332.

A. Induction of apoptosis in HeLa cells

sentation

and

CTL-induced

blocking

IRF-1

expression

lysis in

300. Grimm C, Polterauer S, Natter C, et al.

2014; 184: 358–361. 319. Dhandayuthapani

S,

Marimuthu

P,

Treatment of cervical intraepithelial neo-

310. Arbeit JM, Howley PM, Hanahan D.

via caspase activation by resveratrol and

plasia with topical imiquimod: a random-

Chronic estrogen-induced cervical and

genistein. Journal of Medicinal Food 2013;

ized

controlled

trial.

Obstetrics

&

Gynecology 2012; 120: 152–159. 301. Terlou A, Seters Mv, Kleinjan A, et al. Imiquimod-induced clearance of HPV is associated with normalization of immune

vaginal squamous carcinogenesis in hu-

16: 139–146.

man papillomavirus type 16 transgenic

320. Papazisis KT, Kalemi TG, Zambouli D,

mice. Proceedings of the National Academy

et al. Synergistic effects of protein tyro-

of Sciences 1996; 93: 2930–2935.

sine kinase inhibitor genistein with

311. Riley RR, Duensing S, Brake T, Münger K,

camptothecins against three cell lines

vulvar

Lambert PF, Arbeit JM. Dissection of hu-

in

intraepithelial neoplasia. International Jour-

man papillomavirus E6 and E7 function

255–264.

nal of Cancer 2010; 127: 2831–2840.

in transgenic mouse models of cervical car-

321. Sahin K, Tuzcu M, Basak N, et al. Sensitiza-

cinogenesis. Cancer Research 2003; 63:

tion of cervical cancer cells to cisplatin by

4862–4871.

genistein:

cell

counts

in

usual

type

302. Soong RS, Song L, Trieu J, et al. Toll like receptor

agonist

imiquimod

facilitates

antigen-specific CD8+ T cell accumulation

312. Brake T, Lambert PF. Estrogen contributes

in the genital tract leading to tumor control

to the onset, persistence, and malignant


vitro.

Cancer

the

Letters

role

of

2006;

NF

B

233:

and

Akt/mTOR signaling pathways. Journal of Oncology 2012: 461562.



53

322. Kim SH, Kim SH, Kim YB, Jeon YT, Lee

328. Nguyen HH, Aronchik I, Brar GA, Nguyen

pathway in breast cancer stem-like cells is

SC, Song YS. Genistein inhibits cell growth

DH, Bjeldanes LF, Firestone GL. The die-

required for viability and maintenance.

by modulating various mitogen-activated

tary phytochemical indole-3-carbinol is a

Proceedings of the National Academy of Sci-

protein kinases and AKT in cervical cancer

natural elastase enzymatic inhibitor that

cells. Annals of the New York Academy of Sci-

disrupts cyclin E protein processing. Pro-

334. Bleau A-M, Hambardzumyan D, Ozawa

ences 2009; 1171: 495–500.

ceedings of the National Academy of Sciences

T, et al. PTEN/PI3K/Akt pathway regu-

2008; 105: 19750–19755.

lates the side population phenotype and

323. Hussain A, Harish G, Prabhu SA, et al.

ences 2007; 104: 16158–16163.

Inhibitory effect of genistein on the in-

329. Luo X, Dong Z, Chen Y, Yang L, Lai D. En-

vasive potential of human cervical can-

richment of ovarian cancer stem-like cells

like

cer

is associated with epithelial to mesenchy-

226–235.

cells

via

modulation

of

matrix

transition

through

an

ABCG2 activity in glioma tumor stemcells.

Cell

Stem

Cell

2009;

4:

metalloproteinase-9 and tissue inhibitors

mal

miRNA-

335. Dubrovska A, Elliott J, Salamone RJ,

of matrix metalloproteinase-1 expression.

activated AKT pathway. Cell Proliferation

et al. Combination therapy targeting

Cancer Epidemiology 2012; 36: e387–e393.

2013; 46: 436–446.

both tumor-initiating and differentiated

324. Kim SH, Kim SH, Lee SC, Song YS. In-

330. He K, Xu T, Xu Y, Ring A, Kahn M,

volvement of both extrinsic and intrinsic

Goldkorn A. Cancer cells acquire a drug

Clinical

apoptotic pathways in apoptosis induced

resistant,

5692–5702.

by genistein in human cervical cancer

stem-like phenotype through modulation

336. Hart LS, Dolloff NG, Dicker DT, et al. Hu-

cells. Annals of the New York Academy of Sci-

of the PI3K/Akt/β-catenin/CBP pathway.

man colon cancer stem cells are enriched

ences 2009; 1171: 196–201.

International Journal of Cancer 2014; 134:

by insulin-like growth factor-1 and are

43–54.

sensitive to figitumumab. Cell Cycle 2011;

325. Jin L, Qi M, Chen D-Z, et al. Indole-3-carbi-

highly

tumorigenic,

cancer

cell populations in prostate carcinoma. Cancer

Research

2010;

16:

nol prevents cervical cancer in human

331. Wei Y, Jiang Y, Zou F, et al. Activation of

papilloma virus type 16 (HPV16) trans-

PI3K/Akt pathway by CD133-p85 inter-

genic mice. Cancer Research 1999; 59:

action promotes tumorigenic capacity of

et

3991–3997.

glioma stem cells. Proceedings of the Na-

stem/progenitor cells by PTEN/Akt/β-ca-

tional Academy of Sciences 2013; 110:

tenin signaling. PLoS Biology 2009; 7:

326. Chen D-Z, Qi M, Auborn KJ, Carter TH. Indole-3-carbinol and diindolylmethane induce apoptosis of human cervical can-

6829–6834. 332. Singh

BN,

10: 2331–2338. 337. Korkaya H, Paulson A, Charafe-Jauffret E, al.

Regulation

of

mammary

e1000121. Kumar

D,

Shankar

S,

338. Kreso A, van Galen P, Pedley NM, et al.

HPV16-

Srivastava RK. Rottlerin induces autoph-

Self-renewal as a therapeutic target in hu-

transgenic preneoplastic cervical epithe-

agy which leads to apoptotic cell death

man colorectal cancer. Nature Medicine

lium. The Journal of Nutrition 2001; 131:

through inhibition of PI3K/Akt/mTOR

3294–3302.

pathway in human pancreatic cancer stem

339. Jung YS, Vermeer PD, Vermeer DW, et al.

cells. Biochemical Pharmacology 2012; 84:

CD200: Association with cancer stem cell

1154–1163.

features and response to chemoradiation in

cer

cells

and

in

murine

327. Qi M, Anderson AE, Chen D-Z, Sun S, Auborn KJ. Indole-3-carbinol prevents PTEN loss in cervical cancer in vivo. Molecular Medicine 2005; 11: 59–63.

333. Zhou J, Wulfkuhle J, Zhang H, et al. Activation


of

the

PTEN/mTOR/STAT3

2014; 20: 29–36.

head and neck squamous cell carcinoma. Head & Neck 2014. DOI: 10.1002/hed.23608.


Wnt and GSK3 Signaling Pathways in Bipolar Disorder: Clinical and Therapeutic Implications.

[MET signalling pathway and its therapeutic implications in gastrointestinal cancers].

Signaling Pathways in Leiomyoma: Understanding Pathobiology and Implications for Therapy.

FOXO Signaling Pathways as Therapeutic Targets in Cancer.

HIF-mediated innate immune responses: cell signaling and therapeutic implications.

therapeutic implications.

Phytochemicals modulate carcinogenic signaling pathways in breast and hormone-related cancers.

Akt pathway in Epstein-Barr virus-induced cancers and therapeutic implications.

Kinetics of tumor growth and of cell proliferation in U.R.D.T. cancers: therapeutic implications.

Interaction of autophagy with microRNAs and their potential therapeutic implications in human cancers.

Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications.

Diagnostic and therapeutic implications of new molecular biomarkers in salivary gland cancers.

Differential signaling by protease-activated receptors: implications for therapeutic targeting.

Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications.

Circular RNAs: Regulators of Cancer-Related Signaling Pathways and Potential Diagnostic Biomarkers for Human Cancers.

Correlation between Gene Variants, Signaling Pathways, and Efficacy of Chemotherapy Drugs against Colon Cancers.

Embryological signaling pathways in Barrett's metaplasia development and malignant transformation; mechanisms and therapeutic opportunities.

Bone metastasis-related signaling pathways in breast cancers stratified by estrogen receptor status.

Biliary cancer: intrahepatic cholangiocarcinoma vs. extrahepatic cholangiocarcinoma vs. gallbladder cancers: classification and therapeutic implications.

Non-monotonic dynamics and crosstalk in signaling pathways and their implications for pharmacology.

The role of signaling pathways in cervical cancer and molecular therapeutic targets.

Emerging Role and Therapeutic Implication of Wnt Signaling Pathways in Autoimmune Diseases.

Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer.

Biomarker identification in breast cancer: Beta-adrenergic receptor signaling and pathways to therapeutic response.