7.1 Introduction: Human papillomavirus (HPV) are small epitheliotropic double-stranded DNA viruses that are involved in the etiology of several human cancers. The HPV family includes about 200 categories of which 40 have been isolated from the genital tract. HPVs are divided into two types based on their clinical association: high- and low- risk. The high-risk HPVs are associated with the development of cervical carcinoma in persisted infected females. It is estimated that about 90% of all cervical carcinomas are associated to high-risk HPVs. Of these, HPV-16,-18, and -31 are the most prevalent and commonly linked to lesions that may progress to high-grade intraepithelial neoplasia and ultimately carcinoma. The others which include HPV-6 and -11 are low-risk and associated with benign lesions that rarely progresses to cancer. HPV-16 and -18 accounts for approximately 50% of all cervical cancer cases and cervical cancer is responsible for one-fifth of all cancer-associated deaths among women diagnosed each year making it the second most common cancers among women worldwide. Some of the low-risk maybe transmitted via sexual contact to cause genital condyloma. All the type of the virus shares a common genomic structure (Figure 1) which encodes eight proteins. These include six early E1, E2, E4, E5, E6, and E7, and two late proteins. E5, E6, and E7 oncoproteins of the high-risk strains are considered antiapoptotic oncoproteins which plays significant role in malignant transformation. In addition, E2 and E7 are considered proapoptotic proteins.
Figure 1: Genomic structure of HPV (Source: National Institute of Cancer, 2013)
The importance placed on our understanding of HPV as a contributor to malignant progression resulted in the 2008 Nobel Prize going to Harald zur Hausen for his discovery that high-risk HPV types are associated with the development of cervical cancer. Not every one that is infected with high-risk HPVs develops cancer. Additional genetic alteration is required for the progression to malignant. The HPV oncoproteins E5, E6, and E7 are the primary viral factors associated with the initiation and progression of cervical cancer. They act by overcoming the negative growth instability which is the hallmark of HPV-associated cancers. This chapter will review some of the latest works on the pathogenesis of HPV infection and the role played by these oncoprotein. The life cycle of the virus will be looked at and other important themes such as therapeutic and vaccine intervention will be analyzed.
7.2 HPV Life Cycle: HPV has an unusual life cycle (figure 2). In most viruses, the target cell is infected which results in the production of progeny virus from the same infected cells. In HPV infection, the production of new virions occurs only after cell differentiation. HPVs infects cells found in the basal layer of the stratified squamous epithelial. After infection, the viral genomes are established as extrachromosomal entity or episome. The HPV genomes are small and do not encode polymerase or other enzymes required for viral replication. The virus therefore depends on the host cell replication proteins to mediate viral DNA synthesis. In HPV infection, the suprabasal cells remain active in the cell cycle as they undergo the process of differentiation with a subset of cells entering the S phase to replicate the HPV genome. This process is referred to as amplification. It is followed by capsid protein synthesis, virus assembly, and release.
Figure 2: A typical life cycle of HPV
The proliferative ability of HPVs is controlled by a number of cellular factors, the most important being the members of retinoblastoma (Rb) family. HPV E7 proteins bind to RB family and target them for degradation. This leads to release and activation of E7F transcription factors that promotes the expression of S phase genes. It has been shown that E7 proteins from all HPV types bind Rb family members but the high-risk E7 protein binds with much higher affinity. Efficient binding of Rb by E7 can result in inhibited cell growth and apoptosis through a p53-dependent pathway. By their combined action, high-risk E6 and E7 proteins target these cell cycle regulators to maintain S phase which are competent in differentiation cells and also leads to abrogation of many cell cycle checkpoints. In cells with persistently HPV infection, there is accumulation of cellular mutations overtime with a consequent progression to cancer. The high-risk E5 protein combines rather deadly with E6 and E7 proteins to promote hyperproliferative of infected cells which might facilitate the progression to cancer. From infection to the initiation of cancer, a series of molecular events do take place. The failure of the immune system to clear persistent HPV infection can result in the development of cervical cancer years after harboring the virus. In a precancerous lesions, most of the viral genome persist in episomal form while in the high-grade lesions, the genomes are integrated into the host chromosome with the fragile sites been the most often area of integration. It has been suggested that integration might contribute to cancer progression. Viral E2 protein in the lesions containing HPV reduces early gene expression. Integration of viral DNA is in most case disrupts E2 expression which leads to the deregulated expression of early viral genes including E6 and E7. There is also increased proliferation capacity which is a crucial step in cancer progression. E6 and E7 are essential for maintaining the transformed phenotype. Kadaja et al described an important step in HPV carcinogenesis: coexistence of HPV episome and integrated copies. Replication of integrated sites of origins leads to activation of DNA repair and recombination system. This increases the chance of acquiring cellular mutation, increased genomic instability thereby increasing the opportunity for cancer progression. The various mechanisms associated with cancer development in HPV infection will be dealt with later.
7.3 E6, E5, and E7 Oncoproteins
7.3.1 E6 oncoprotein: The HPV E6 oncoprotein is a relatively small protein which was long recognized as a potent oncogene and ultimately associated with events that result in the malignant conversion of virally infected cells. Analysis of HPV-16 E6 oncoprotein showed that is made of 150 amino acids, containing two CX2C-X29-CX2C zinc-like fingers which are joined by interdomain linkers of 36 amino acids. In the high-risk E6 genes, shortened E6 can inactivate the function of full length E6 by binding to the interface of the C- and N- terminal halves of E6 proteins. E6 oncoprotein does not have any enzymatic activities but most of its major activities are triggered by protein-protein interactions. The first protein that interacts with E6 is E6-associated protein (E6AP) which is an ubiquitin ligase. E6, E6AP and target protein form a complex that results in the ubiquitination of the target protein with a subsequent proteasome-mediated degradation. One of the main targets of E6 is p53, a key signaling coordinator in the cell after genotoxic or cytotoxic stress. E6 protein binds to p53 with the assistance of E6AP and stop p53 from inducing apoptosis by targeting it for degradation through ubiquitin-pathway. E6 oncoprotein is involved in two pathways that are associated with apoptosis: p53 inactivation and blocking apoptosis. In the first instance, p53 inactivation may trigger the E6-induced apoptosis inhibition. The most important mechanism for p53 activation by high-risk HPV is induction of p53 degradation via the ubiquitin-proteasome pathway. Additionally, E6 protein in high-risk HPV can inhibit p53 activation by blocking the alternative reading frame p14 pathway and by interacting with a histone acetlytransferase, hAD3. Secondly, inhibition of apoptosis maybe triggered by E6 oncoprotein. It has been found that p53-independent apoptosis is also capable of eliminating abnormal cells while E6 is capable of blocking apoptosis in cell and mice lacking p53. There are two major apoptotic pathways: intrinsic and extrinsic pathways. E6 is able to disturb these pathways and prevent cell death under endogenous and exogenous stress.
7.3.2 E5 proteins: E5 is the smallest among HPV oncoproteins and its ORF has been classified into four different groups based on the different clinical manifestations, especially with the potential for oncogenesis: alpha, beta, gamma, and delta. In HPV-16, E5 is encoded by 83 amino acids. The E5 ORF is absent in the genome of many HPVs which indicate that the protein is not essential for the life cycle of these viruses but it gives an added value to favor infection and transformation. HPVs are thought to use leaky ribosome-scanning mechanism to translate protein from polycistromic mRNA. Little E5 protein is mostly likely to be synthesized from these transcripts. However on epithelial –cell differentiation, E5 is expressed on the second ORFs of late transcript. The suggestion is E5 is likely synthesized mostly in differentiating suprabasal epithelial cells. Detection of E5 protein is difficult due to its extreme hydrophobicity, membrane localization and low levels of expression. Change et al reported that since HR E5 gene is able to integrate into human genome during malignant progression, the E5 gene is rarely found in cervical tumors. HPV E5 inhibits death receptor-mediated apoptosis in human keratinocytes and capable of downregulating the total amount of Fas receptor and decreasing Fas location. It also alters the formation of DISC induced by TRAIL. This means E5 can impair Fas ligand (FasL) and TRAIL-mediated apoptosis. HPV-16 E5 protein has been found to suppress three main proteins in the ER stress pathway. These include cyclooxygenase-2 (COX-2), X-box binding protein-1 (XBP-1) and inositol-requiring enzyme-1α (IRE1α). The downregulation of these is beneficial for viral replication and persistence. In cervical cancer cells, EP4 protein can be activated by HPV-16 E5, which activates protein-kinase A which is responsible for antiapoptotic effect. Additionally, activating EP4 can enhance the expression of VEGF with a resultant tumor immortalization in cervical carcinoma. The transforming activity of E5 has been described in a number of cell types and assays.
7.3.3 E7 proteins: Oncoprotein E7 is a small acidic polypeptide encoding for about 100 amino acids. It shares functional similarities with other viral oncoproteins especially adenoviral E1A and SV40 large T antigen, both in primary sequence, transactivation and transformation properties. Based on its amino acid sequence, E7 can be separated into three conserve regions just like in adenovirus E1A as: CR1, CR2, and CR3. The CR2 and CR3 region of HPV E7 share the same sequence with the corresponding regions of adenovirus E1A and SV40 large T antigen, including a strictly conserved CXCEX motif which mediates high affinity for pRb. The CR3 region of E7 is made of 2 CXXC motifs which are separated by 29 or 30 residues, forming a zinc binding domain. Studies have shown that HPV C3 mediates protein dimerization and mediates direct interaction with several E7-interacting proteins. The E7 CR3 region can also mediate inactivation of the cyclin-dependent kinase inhibitorsp27 and p21 as well as several transcription factors that are implicated in HPV-associated oncogenesis including TATA-box-binding protein (TBP), the transcription factor E2F, and p300/CBP-associated factor (P/CAF). The HPV-16 E7 oncoprotein induces p53-depedent and independent apoptosis. It mediates cell transformation in part by binding to the human pRb tumor suppressor protein and E2F transcription factor which leads to dissociation of pRb from E2F transcription factor and the prevents cell progression to S phase of the cell cycle. This activity is mediated by Leu-X-Cys-X-Glu (LXCXE) motif and CR3 zinc binding domain of the E7 protein. E7 leads to the antiapoptotic pRb degradation through a mechanism involving association with and reprogramming of cullin 2 ubiquitin ligase complex. It has been shown that the C-terminal of E7 oncoprotein is made of low-affinity pRb binding site which interacts with pRb. Other pathways have been identified that triggers apoptosis with the involvement of E7 oncoprotein. For example, E7 protein activates apoptosis in NIH3T3 cell through a conserved LXCXE motif in the second chromodomain (CD2) of the E7 structure binding to pRb. Another pathway involves E7 and p21 forming a complex which activates Cathepsin B, a known apoptotic mediator. Another pathway involves E7 oncoprotein inducing TRAIL and TNF-α-induced apoptosis in primary human keratinocytes. In addition, it has been reported that E7-induced apoptosis is associated with interaction between E7 and E2F1. The complex is able to trigger E2F1- driven transcription contributing to increased apoptosis. Other mechanisms of inhibiting apoptosis and cytokine-mediated cell deatj by HPV E7 oncoprotein have been described. It was reported that HPV-16 E7 interact with and abrogate the growth –inhibitory activities of cyclin-dependent kinase inhibitor (CKI) sp21 and CKI sp27 as well as abrogating TGF-β-mediated growth inhibition. Another study reported that HPV-16 E7 protein inhibits TNF-α-mediated apoptosis in normal human fibroblasts by up regulating the expression of inhibition of apoptosis (IAP) protein, c-IAP2. This is aided by suppression of caspase 8 activation. Siva-1 is a proapoptotic cellular factor capable of binding to the antiapoptotic protein Bc1-X2. When HPV E7 interferes with the binding of Siva-1 and Bc1-XL, the released Bc1-XL is able to exert its antiapoptotic effect. Finally, it has been reported that HR or LR HPV are able to interact with the 600kDa pRb-associated factor, p600. Therefore the conjugation of E7 and p600 can protect the detached cells from apoptosis thereby contributing to viral transformation.
7.4 Biomarkers of HPV –associated carcinomas: A biomarker is a substance or process that is indicative of the presence of cancer in the body. The US National Institute of Health’s (NIH) Working Group and the Biomarkers Consortium defined biomarkers as “a characteristic that is objectively measured as an indicator of normal biologic process, pathogen process, or a pharmacological response to a therapeutic intervention”. The NIH’s National Cancer Institute (NCI) described biomarkers as “biological molecules found in blood, other body fluids, or tissues that is a sign of a normal process,, or a condition or disease”. The World Health Organization defines biomarkers as any substance, structure, or process that can be measured in the body or its product and influences or predicts the incidence of outcome or disease. Biomarkers has also been defined a measureable phenotypic parameter that characterize an organ, state of health or disease, or a response to a particular therapeutic intervention. Similar biomarkers can also be defined based on physical, chemical. Biological entities which are accessible in the body matrices and measureable in body fluids or cells. Biomarkers are sometimes referred to as molecular markers and signature molecules. A cancer biomarker may be a molecule secreted by a tumor or a specific response of the body to the presence of cancer. Genetic, epigenetic, proteomic, glycomic, and imaging biomarkers can be used for cancer diagnosis, prognosis, and epidemiology. Ideally, such biomarkers should be assayed in non-invasively collected biofluids such as blood or serum. Several biomarkers have been identified in HPV infection and carcinomas which identify specific stages in the natural history of HPV infection and cancer progression. They include the presence of viral nucleic acids, viral proteins, or alteration of cellular factors induced by viral oncoproteins. In cervical cancer,, numerous assays have been developed to detect nucleic acids of oncogenic and non-oncogenic HPVs in cervical cancer. The advantage of using HPV testing is that it is highly sensitive with a high predictive values due to the fact that the absence of carcinogenic HPV indicates an extremely risk of CIN3 or cancer, and a long protection compared to cytology because the risk of CINs and cancer remains very low up to 5 years after a negative HPV test. There is one concern: the low specific of HPV assay because they cannot discriminate between transient and persistent HPV infections. Nevertheless, data from number of studies has shown that high risk HPV testing is highly sensitive and specific for detecting CIN2. There are available a number of HPV assays and are grouped into two categories based on: 1. Their ability to identify a pool of HR HPVs types with or without genotyping of the most common high risks ones, or 2. To detect a broad spectrum of oncogenic and nononcogenic HPVs along with individual genotyping. The assays available include HPV DNA assays of which four has been approved by FDA: 1. Hybrid capture 2 (Qiagen), detecting 13 HR HPVs; 2. Cervista HPV (Hologic), targeting 14 HR HPVs; 3. Cervista HPV 16/18 (Hologic), specifically designed for identifying HPV 16 and 18; 4. Cobes 480 HPV (Roche Diagnostics), targeting 14 HR HPVs. HPV RNAs assay have been developed that detect viral mRNA encoded by E6 and E7 oncoproteins. Four assays are currently available for detecting HPV E6 mRNA in cervical samples. These are: APTIMA (Gen Probe) and Onco Tech (Incell DX) assays which are based on RT and PCR techniques to detect E6/E7 from 14 and 13 HR HPV genotypes respectively. PreTech HPV- Proofer ( Norchip) and NucliSENS EasQ (Biomerieux) both rely on nucleic acid sequence-based amplification (NASBA). They are able to detect E6/E7 transcripts from five of the most common HR HPV types in cervical carcinoma (HPV 16, 18, 31, 33, and 45). Two HPV protein assays are also available for detecting protein level in cervical cell exfoliates. These are OncoE6 (Arbor Vita corporation) and Cyto active (Cytoimmune Diagnostic). The oncoE6 is used to identify the E6 protein encoded by HPV 16, 18, and 45. The cyto active assays are designed to measure the loss of L1 expression which is a predictive marker of progressive lesions. In addition to these biomarkers, cellular biomarkers have been developed such as P161NK4a assay which is important tool for the improvement of the diagnostic accuracy, reliability, and quality in histopathology analysis of cervical lesion and BD Pro ExCTM marker assay which has a higher sensitivity for detecting women with low SIL than women with high SIL.
The E6 protein promote transcription of telomerase reverse transcriptase (TERT) which stabilizes and repairs repeated DNA sequences at the telomere end of chromosomes. Gains of chromosome 3q which contain the sequence for the telomerase RNA component (TERC) and gains of chromosome 5p containing TERT gene are associated with CIN2 or worse in cervical tissue biopsies. Therefore evaluation of chromosome 3q and 5p with FISH and multiplex ligation-dependent probe amplification (MLPA) may be a useful marker for diagnosing progressing lesions. But the analysis of TERT and TERC copy number increase will be difficult in cytology samples as a result of the presence of predominantly normal cells in the samples. One novel indicator of HPV carcinogenesis is miRNAs. Several miRNAs such as miR-9, miR-21, miR-27a, miR-127, etc have been described in the dysregulation of cervical cancer and other HPV-associated carcinomas. Riberio et al reported that in 114 women with different cervical lesions, miR34a expression seems to correlate with invasive cervical cancer while miR-125b expression significantly changed with the different cervical lesions. Li et al also reported that among 171 women with CIN, miR-218 levels were lower in patients with HR HPV than those with LR or intermediate HPVs. Wang et al showed that the expression of miR-375 in 170 cervical cancer tissues significantly reduced compared to 68 normal tissues, suggesting that downregulation of miR-375 could be associated with cervical cancer. Malta et al showed that Let-7c, a microRNAs precursor significantly changed in exfoliated cervical cells from women with cervical intraepithelial lesions. Other molecules that have the potential to function as predictive markers of cervical cancer and other HPV-associated carcinomas include Yes-associated protein (YAP), Laminin receptor, E-cadherin, etc. As more alternative biomarkers result in better diagnosis and management of HPV-associated carcinomas, it is important more studies, both basic and clinical; to identify and optimize more and available biomarkers.
7.5 Anti-Oncoprotein Agents, novel approaches: Current treatment strategy for HPV-associated carcinomas involves use of radiotherapy with cisplatin-based chemotherapy, surgery while a limited success has been achieved with immune modulators such as IFN. In addition prevention of HPV infection by vaccination and challenge of established HPV infections by immune therapy is still been investigated. With the radiotherapy, it involves radiotherapy with cisplatin-based chemotherapy. Statistically, about half a million mortality associated with cervical cancer are reported yearly and a much higher number of patients are exposed to noninvasive disease or genital warts. This means we can conclude that from this data that current treatment of HPV-associated carcinomas is not adequate. Development of alternative treatments options is needed. With advances in molecular biology, it is now possible to target specific HPV proteins involved in virulence. A number of novel ideas have been investigated. This can be done by optimization some existing drugs or identifying new compounds that can inhibit proteins in the virus’s life cycle. With the former, a number of novel optimization studies have been undertaken. For example, Bortezomib is a proteasome inhibitor while suberoylanilide hydroxamic acid (SAHA, also called Vorinostat) is a histone deacetylase inhibitor which is recognized as potential chemotherapeutic drug. Both drugs have been approved by the FDA for the treatment of cutaneous T cell lymphoma and multiple myeloma/ mantle cell lymphoma. In a study, Huang et al showed that combination of bortezomib and SAHA elicited potent antitumor TC1-tumor bearing mice which led to tumor-specific immunity by antigen-specific CD8+ T cells than treatment with either drug alone. They therefore concluded that the study can serve as the foundation for future application of both drugs for the treatment of cervical cancer. Earlier, Lin et al examined the effects of SAHA with bortezomib on tumor cell apoptosis. They found that the combination elicited synergistic killing of HPV-expressing cervical cancer cell lines and the combination treatment diminished tumor growth of HeLa xenografts more effectively than either drug alone. In a systematic review study, this author concluded the IM IFN in combination with 5% imiquimod has effect on HPV-associated cervical cancer but study is needed to evaluate this combination in clinical trial. Addition of platin-based chemotherapy to radiotherapy has increased 5-year survival of advanced stage of cervical cancer patients. However there have been reported cases of treatment failure. One of the factors associated with treatment failure is the ability of the tumor cells to repair chemotherapy-induced DNA damage. Therefore is has been suggested that sensitization of tumor cells for chemotherapy via inhibition of the DNA damage response (DDR) is a novel strategy to improve therapeutic effect. Cervical carcinogenesis involving HPV infection inactivates part of the DNA damage response. Therefore HPV-mediated partial inactivation of DDR is a novel therapeutic target. Another approach is blocking angiogenesis and this strategy has been reported in a number of studies. Bevacizumab is a recombinant humanized monoclonal antibody to VEGF. It has been shown to have a role in the treatment of many cancers. It has been approved by the US FDA for advance cervical cancer. This has opened the door for future studies on this anti-angiogenesis as backbone of chemotherapy via targeting additional pathways. But patients who were treated with concurrent bevacizumab and chemotherapy developed fatigue, anorexia, hypertension, hyperglycemia, hypomagnesaemia, headache, weight loss, and urinary tract infection. A more serious side effect, gastrointestinal perforation, was reported in 3.2% of the patients treated with the drug. The focus of this review is on potential anti-oncoproteins for therapeutic intervention as part of the new drug model. As explained earlier, strategies to interfere with oncoprotein-induced p53-degradation in order to restore the function of p53 is a promising therapeutic option for cervical cancer. A study showed that as a consequence of HPV 16 E6-induced p53 inactivation, two kinases, serum and glucocorticoid regulated kinase 2 (SGK2) and p21-activated kinase 3 (PAK3) played an essential role in cells proliferation and viability. When cells lost both p53 and either SGK2 or PAK3, cell death occurred. The kinase did not show similar function in non-infected human foreskin keratinocytes expressing wild-type p53. This means a novel therapeutic strategy may be developed based on synthetic lethal interaction between loss of p53 and drugs targeting either SGK2 or PAK3 in HPV-associated cervical cancer. The zinc domain is critical for the function of E6 oncoprotein. This can be a novel target. In a study, Beerheide et al found that the compound 4, 4’-dithiodimorpholine selectively inhibited cell viability and induced higher levels of p53 protein in tumorigenic HPV-containing cells. This compound, the authors concluded can potentially interfere with the biology and pathology of HPV. Dithiocarbamates (DTCs) have been reported to exhibit broad spectrum of antitumor activities. In a study Li et al provided evidence that DTC1 suppressed both expression of E6 mRNA and E6 oncoprotein but had no effect on the expression of E7 mRNA and protein in HPV-18. DTC1 may therefore serve as a novel chemotherapeutic agent for the treatment of cervical cancer and potential anti-HPV candidate. DTC1 has a number of abilities. It could inhibit proliferation; induce apoptosis in HeLa cells by activating caspase-3,-6, and -9. In addition, it can decrease the levels of Bcl-2, and Bcl-XL. Furthermore it induced HeLa cell apoptosis through p53-dependent pathway. Duplex formation between the branch point-binding region (BBR) of U2 snRNA and the branch point sequence (BPS) in the intron is essential for splicing. Several lines of evidence indicate that splicing is functionally couple to transcription. Both BBR and BPS interact with the U2 small nuclear ribonucleoprotein (snRNP)-associated complex. This is interaction which is targeted by the anti-tumor drug E7107 as reported by Folco et al. E7107 blocks spliceosome assemble by inhibiting the binding of U2 snRNP to pre-mRNA. The compound does not have any effect on U2 snRNP integrity but it abolishes ATP-dependent conformational change in U2 snRNP that exposes BBR. More studies is needed to understand the effect of E7 on U2 RNP which will contribute to the establishment of the therapeutic potential of E7107 which will lead to the development of second-generation derivatives. A number of plant compounds have also been described. Wogonin is extracted from Scutellaria baicalensis and is known as a benzodiazepine receptor ligand with anxiolytic effect. Studies have shown that it modulates angiogenesis, proliferation, invasion, and tumor progression in various cancer tissues. A study by Kim et al showed that wogonin was cytotoxic to HPV-16 positive cervical cancer and that it induced apoptosis by suppressing the expression of E6 and E7 viral oncogenes. It was found that it also modulate the mitochondrial membrane potential and the expression of pro and anti-apoptotic factors such as Bax and Bcl-2. Finally, Luteolin is flavonoids extracted from a number of plants with recognized anticancer, anti-inflammatory, and anti-oxidative properties, inhibits angiogenic process, and modulate multidrug resistance. A study by Ham et al showed that Luteolin had a significant dose-dependent cytotoxic effect only in HPV-positive cells in comparison with HPV-negative cervical cancer cells. Expression levels of HPV oncogenes E6 and E7 were suppressed while those of related factors pRb and p53 were recovered and E2F5 was increased by Luteolin treatment. Furthermore, it Luteolin enhanced the expression of death receptors and death receptors downstream factors such as Fas/FasL, DR5/TRAIL and FADD in HeLa cells, and activated caspase cascades. Luteolin also induced mitochondria membrane potential collapse and cytochrome release and inhibits Scl-2 and Bcl-xL expression. The authors concluded that Luteolin exerts anticarcinogenic ability through inhibition of E6 and E7 expression and apoptosis by activating intrinsic and extrinsic pathways.
Variety of promising techniques are now available to aid in the search for new compounds for the development of novel anti-oncoprotein drugs for the treatment of HPV-associated cancers. Two promising methodologies will be dealt with in chapters 14 and 15. The importance of High -throughput screening (HTS) will be discussed briefly here. HTS is a specific development in laboratory automation to collect large amount of experimental data in a relatively short time to test hundreds and thousands of compounds per day. It has been shown that an HTS process can allow up to 1,000 times faster screening (100 million reactions in 10 hours) at 1-millionth a cost than conventional techniques using drop-based micro fluidics. Through this one can rapidly identify active compounds, antibodies, or genes that modulate a particular biomolecular pathway. This provides starting point for drug design and understanding the interaction and role of a particular gene or pathway in pathogen-host interaction. With oncoviral infections, HTS screening could lead to the design of small molecules that can inhibit directly the oncogenic proteins that are mutated or overexpressed in specific tumor cell types. By targeting specific molecules defect found within the tumor cells, HTS may ultimately yield therapies tailored to each tumor’s specific makeup. This methodology resulted in the development of Gleevec (Matinib), an inhibitor of the breakpoint cluster region-abelsen kinase (BCR-ABL) oncoprotein found in Philadelphia chromosome-positive chronic myelogenous leukemia and Herceptin (trastuzumab), a monoclonal antibody targeted against oncoprotein found in metastatic breast cancers. A strategy that can be adopted in HTS involves searching for genotype-selective anti-tumors agents that become cidal to tumor cells only in the presence of specific oncoprotein such as E6 or in the absence of specific tumor suppressors. Such compounds might target oncoproteins directly or might target other critical proteins involved oncoprotein-associated signal network. The following compounds have been reported to possess synthetic cidal abilities: 1. Rapamycin analog CCI-779 in myeloma cells lacking PTEN, 2. Gleevac in BCR-ABL –transformed cells and 3. Benzoquinazolines and thiazoloinidazole for influenza viruses. Stockwell et al used an assay format they termed cytoblot to identify a number of less-characterized compounds. They showed that cytoblots can be used for high-throughput screening of small molecules in cell-based assay. A number of HTS screening strategies are available. These include cell-based assay, cytopathic effect (CPE)-based assay, and ATP/luminescence assay, etc. HTS is used to identify lead compounds for a wide range of therapeutic areas. Typically, a single data read is used to determine whether the test compound interacts with the target of interest. The result of HTS activity leads to the compound been grouped into either a hit if the target shows activation above a specified threshold and non-hits is it falls below the threshold. The assay threshold typically depends on simple signal-to noise activities. Cell-based assay HTS screening can also be used for absorption, distribution, metabolism, elimination, and toxicity (ADMET) in the early stage of drug discovery. The CPE-based antiviral HTS assay involves cell-based screen for potential antiviral compounds that measures the CPE induced by the viral infection in a cell to measure cell viability. With luminescence assay, a luciferase gene is engineered into a Replicon or full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. With the availability of very promising screening assay, more novel ideas should be developed to facilitate the development of the “magic bullet” for HPV-associated carcinomas.
7.6 HPV Vaccines: So far two prophylactic HPV vaccines are in use: Gardasil, a quadrivalent HPV 16/18/6/11 virus-like vaccine developed by Merck and Co, Inc. (NJ,USA) and Cervarix, a bivalent HPV 16/18 particles from GSK plc (London, UK). They have been found to be efficient in preventing HR HPV infections and minimize the consequences of HPV-associated diseases. But these vaccines are only effective in girls from 11 to 12 years as recommended by the FDA, without history of past history of HPV infection and not shown therapeutic effect against current infection or associated lesions. From these, it can be argue that a large population will remain at risk of HPV infection. This makes the development of therapeutic vaccine against HR-HPV a global health priority; especially of HPV-16 which is responsible for 46% to 65% of squamous cell carcinomas worldwide. In addition, HPV-58 which is the third most common virus in cervical cancer is Asia need to be a point of focus for therapeutic vaccine. This section will review some of the novel ideas been utilized for the development of HPV therapeutic vaccine. A review studies provided an overview of the efficacy and clinical effectiveness of a bivalent (HPV 16, 18), quadrivalent (HPV 6,11,16,18) and 9v HPV (HPV 6, 11, 16, 18, 31, 33, 45,52, 58) vaccines reported that all the three vaccine showed some high efficacy in the prevention of vaccines-specific HPV-type infection and associate high-grade cervical dysplasia in HPV-naïve women. An early clinical effectiveness data for the bivalent and quadrivalent vaccine showed reduced rates of HPV 16 and 18 prevalence in vaccinated cohorts. In addition, the bivalent vaccine exhibited cross-protection to non-vaccine HPV types. However there is no clinical effectiveness data for 9v HPV vaccine. Apo Immune has developed a novel HPV vaccine, ApoVax104-HPV which comprises Chimeric molecule containing extracellular domain of costimulatory 4-1 BBL fused C-terminus to core streptavidin (ApoVax104) and biotinylated HPV-16 E7 oncoprotein as a TAA conjugated to the Chimeric protein via biotin/streptavidin interaction. A phase I studies showed that ApoVax104 component of the vaccine targeted conjugated antigen into DCs constitutively expressing the 4-1BB receptor which activated the DCs for antigen uptake and presentation which led to initiation of adaptive immunity. It also directly worked on CD4+ and CD8+ T effector (Teff) cells to augment the adaptive immunity and most importantly, it overcame the suppressive function of CD4+CD25+FoxP3+T regulatory (Treg) cell. The pleotropic effects of 4-1BL on innate, adaptive and regulatory immunity gives it some advantage over novel vaccines which are being developed. Wang et al in a study modified HPV58 E6 and E7 oncogenes to eliminate their oncogenic potential then constructed a recombinant DNA vaccine that co-expressed the sig-HPV58ME6E7-Fc-GPI fusion antigen plus Granulocyte-Macrophage colony stimulating factor (GM-CSF) and a B7.1 serving as molecular adjuvant (PVAXI-HPV58mE6E7FcGB) for the treatment of HPV58-positive cancer. Mutant mice were challenged by HPV58 E6E7-expressing B/6-HPV58 E6E7 cells. This was followed by immunization with the test vaccine on days 7, 14, and 21 after the tumor challenge. They reported that the test vaccine elicited varying levels of IFN-1sgdB T-cell immune response. They therefore concluded that the test vaccine efficiently generated cellular immunity and antitumor efficacy in the immunized mice. All these novel strategies are still at hypothetical phase. Large populations studies are needed to estimate these vaccines. However HPV vaccine carries unique challenges. Three issues are highly essential: Acceptability, dose adherence, and knowledge of healthcare providers. Vaccine acceptability is a crucial factor in vaccine uptake. Belief and willingness to be vaccinated are crucial factors. A study by Hoque and Van Hal aimed at investigating the acceptability of HPV vaccine among educated people in South Africa found that month 146 MBA students, majority of them (74%) have heard of cervical cancer but only 26.2% had heard of HPV. After reading some information on cervical cancer and HPV, the intention to have their daughter vaccinated increased from 88% to 97.2%. This can be attributed to fears of the disease. Those who did not want their daughters vaccinated attributed their decision to the safety of the vaccine. Gilbert et al reported that among 296 heterosexual men and 312 gay and bisexual men in the US, more gay and bisexual men were willing to receive HPV vaccine than heterosexual men. Gay and bisexual men reported of greater awareness of HPV vaccine, perceived worry of HPV-associated diseases, perceived effectiveness of HPV vaccine, and anticipated regret if they declined vaccination and later developed HPV-associated diseases than heterosexual men. This means novel intervention strategies for heterosexual group may be needed. A systematic studies of 14 unique studies representing 10 sub-Saharan Africa countries reported that acceptability of HPV vaccine for females was high but vaccine-related awareness and knowledge were low. In Ghana, it was found that of the 204 women, aged 18-65 who took part in a survey, 94% were willing to vaccinate themselves or allow their daughters to be vaccinated. Side effects and safety of needles were some of the prevalent concern. In the North of Nigeria which has being plagued by rejection of polio vaccine, Iliyasu et al reported that among 375 female university students, 74% were willing to accept HPV vaccination. Another study in Nigeria showed that 70% of 201 mothers were willing to accept vaccination for their children. The 30% stated sexual promiscuity as reason for refusing HPV vaccination. Dose adherence is also essential. Without adhering to the vaccination schedule, the success of any program will be thin. In the US, HPV vaccine has been recommended for use in girls and young women since 2007 but HPV vaccine uptake is low. A study by Liu et al reported that among 378,484 females aged 9-26 years, only 29.4% completed HPV vaccination. Another study by Hirth et al in the US involving 271,976 female found that females aged 13 years to 18 years; 19 years to 26 years, and ≥ 27 years were less likely than those 9 years to 12 years to complete their HPV vaccine schedule. Even among men, adherence is a problem. Hirth et al found that among 514 males who initiated HPV vaccination between 2006 and May of 2009, only 21% completed all 3 vaccine doe within 12 months and completion decreased over time. More of the studies reviewed showed high acceptance rate with few rejections. Reasons for refusing HPV vaccines include safety concern and its efficacy, and belief. It is therefore imperative that HPV campaign should address gaps in knowledge regarding HPV, genital warts, and cervical cancer. It should attend to concern about vaccine safety and efficacy. In addition, strategies should be developed to address the issue of social stigma surrounding HPV vaccine as well as promoting families and partners support for women who decide to be vaccinated. In the words of Larson : the world must accept that HPV vaccine is safe!
References
1. ACT Against cancer: NCI announces potential HPV-related biomarker and spreader of clinical trials on www.uacancer.com/home/category/hpv 6/18/2013 (Accessed on 10th December 2015).
2. Agrestia JJ, et al (2010): Ultrahigh-throughput screening in drop—based micro fluidics for directed evolution, PNAS USA; 107: 4004-4009.
3. Alonso LG, et al (2004): The HPV 16 E7 oncoprotein self assemble into defined spherical oligomer, Biochemistry; 43: 3310-3317.
4. An WF, Tolliday N (2010): Cell-based assays for high-throughput screening, Mol Biotechnology; 45: 180-186.
5. Arbeit JM, et al (1996): Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus transgenic mice, PNAS USA: 93 2930-2935.
6. Arbyn M, et al (2012): Evidence regarding human papillomavirus testing in secondary prevention of cervical cancer, Vaccine; 30: F88-F99.
7. Arbyn M et al (2013): The APTIMA HPV assay versus the Hybrid capture test in triage women with ASC-US or LSIL cervical cytology: a meta-analysis of the diagnostic accuracy, Int J of Cancer; 132: 101-108.
8. Awakumv N, Torchia J, Mymryk JS (2003): Interaction of the HPV E7 protein with the pCAF acetyltransferase, Oncogene; 22:3833-3841.
9. Baker CC, et al (1987): Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines, J Virol; 61: 962-971.
10. Baldwin A, et al (2010): Kinase requirement in human cells. V. synthetic lethal interaction between p53 and the protein kinase SGK2 and PAK3, PNAS USA; 107: 12463-12468.
11. Bao YP, et al, ACCPAB Members (2003): Human papillomavirus type distribution in women from Asia: meta-analysis, Int J Gynecol Cancer; 18: 71-79.
12. Beeheide W, Bernard H-U, et al (1999): Potential drugs against cervical cancer: zinc-ejecting inhibitors of the human papillomavirus type 16 E6 oncoprotein, JNCI; 91:1211-1220.
13. Bentley D (1999): Coupling RNA polymerase II transcription with pre-mRNA processing, Curr Opin Cell Biol; 11: 347-351.
14. Bentley D (2002): The mRNA assembly line: Transcription and processing machines in the same factory, Curr Opin Cell Biol; 14: 336-342.
15. Boucher J, et al (2009): Evaluation of p161NK4a minichromosom maintenance protein 2, DNA topoisomerase IIα, ProEx C, and p141NK4a/ProEx in cervical squamous intraepithelial lesions, Human Pathology; 40:904-905.
16. Branca M, et al (2006): Relationship of up-regulation of 67-kd laminin receptor of cervical intraepithelial neoplasia and to high-risk HPV types and prognosis in cervical cancer, Acta Cytol; 50: 6-15.
17. Branca M, et al (2006): Down-regulation of E-cadherin is closely associated with progression of cervical intraepithelial neoplasia (CIN) or disease outcome in cervical cancer, Eur J Gynaecol Oral; 27: 215-223.
18. Bravo IG, Alonso A (2004): Mucosal human papillomaviruses encode four different E5 proteins whose chemistry and phylogeny correlates with malignant or benign growth, J Virol; 78: 13613-13626.
19. Brehma A, et al (1999): The E7 oncoprotein associates with Miz and histone deacytylase acitivity to promote cell growth, EMBO J; 18: 2449-2458.
20. Capdeville R, et al (2002): Glivec (STI571, IMATINIB), a rationally developed, targeted anticancer drug, Nat Rev Drug Discove; 1:493-502.
21. Castle RE, et al (2010): Relationship of atypical glandular cell cytology, age, and human papillomavirus detection to cervical and endometrial cancer risks, Obs & Gynecol; 115:243-248.
22. Cheng S, Schmidt-Grimminger DC, et al (1995): Differentiation –dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiation keratinocytes, Gene Dev; 9: 2335-2349.
23. Clements A, et al (2000): Oligomerization properties of the viral oncoprotein adenovirus E1A and human papillomavirus E7 and their complexes with the retinoblastoma protein, Biochemistry; 26: 289-293.
24. Clemens KE, et al 1985): Dimerization f the human papillomavirus oncoprotein in vivo, Virology; 214: 289-293.
25. Clifford GM, Smith JS, et al (2003): Human papillomavirus types in invasive cervical cancer worldwide: a Meta analysis, Br J Cancer; 88: 63-73.
26. Coleman MA, et al (2011): HPV vaccine acceptability in Ghana West Africa, Vaccine; 29: 3945-3950.
27. Cunningham MI, Davison C, Aronson KJ (2014): HPV vaccine acceptability in Africa: A systematic review; Science; 69: 274-278.
28. Dahiya A, et al (2000): Role of the LXCXE binding site in Rb function, Mol Cell Biol; 20: 6799-6805.
29. Das R, Dufu K, et al (2006): Functional coupling of RNAR II transcription to spliceosome assemble, Genes & Dev; 20: 1100-1109.
30. Depuydt CE, et al (2011): BD-Pro ExC as adjunct molecular marker for improved detection of CIN+ after primary screening, Cancer Epidemiology & Prevention; 20: 628-637.
31. Deunsing S, Munger K (2004): Mechanism of genomic instability in human cancers: insight from studies with human papillomavirus oncoproteins, Int J Cancer; 109:157-162.
32. de Villiers EM, et al (2004): Classification of papillomavirus, Virology; 324:17-27.
33. Druker BJ, et al (1996): Effects of a selective inhibitor of the Ab1 tyrosine kinase on the growth of Bcr- Ab1 positive cells, Nat Medicine; 2:561-566.
34. Dyson N, et al (1989): The human papillomavirus -16 E7 oncoprotein is able to bind to the retinoblastoma gene product, Science; 243: 934-937.
35. Eichten A, et al (2004): Molecular pathways executing the ‘trophic sentinel’ response in HPV-16 E7 expressing normal human diphoid fibroblasts upon growth factor deprivation, Virology; 319: 81-93.
36. Ezeanochie MC, Olagbuji BN (2014): Human papillomavirus: determinant of acceptability by mothers for adolescents in Nigera, Afr J Reprod Health; 18: 154-158
37. Fisher CM, Schefter T (2015): Profile of bevacizumab and its potential in the treatment of cervical cancer, Onco Target; 8: 3425-3431.
38. Folcon EG, Coil KE, Reed R (2011): The anti-tumor drug E7107 reveals an essential role for SF3b in remodeling U2 snRNP to expose the branch point-binding region, Genes & Dev; 25: 440-444.
39. Francis DA, et al (2000): Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells, J virol; 74: 2671-2686.
40. Gadduci H, et al (2013): Tissue biomarkers as prognostic variables of cervical cancer, Crit Rev in Oncol & Hematol; 86: 104-129.
41. Gage JR, et al (1990): The E7 protein of the non oncogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties, J virol; 64:723-730.
42. Garrett TO, Duerksen-Hughes PJ (2006): Modulation of apoptosis by human papillomavirus (HPV) oncoproteins, Arch Virol; 151: 2321-2335.
43. Gilbert P, et al (2010: HPV vaccine acceptability in heterosexual, gay, and bisexual men, Am J Men Health; 5: 4297-435.
44. Gocze K, et al (2015): MicroRNA expression in HPV-induced cervical dysplasia and cancer, Anticancer Res; 35: 523-530.
45. Gong EY, et al (2013): Development of a high-throughput antiviral screening assay for screening inhibitors of Chikungunya virus and generation of drug-resistant mutation in cultured cells, Methods Mol Biol; 1030: 429-438.
46. Goodwin EC, DiMaio D (2000): Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cell carries the orderly reactivation of dormant tumor suppression pathways, PNAS USA; 97: 12513-12518.
47. Griesser H, et al (2009): HPV vaccine protein L1 predicts disease outcome of high-risk HPV+ early squamous dysplastic lesions, The America J of Clinical Pathol; 132:840-848.
48. Ham S, K Ki H, et al (2014): Luteolin induces intrinsic apoptosis via inhibition of E6/E7 oncogenes and activation of extrinsic and intrinsic pathways in HPV-18-associated cells, Oncology Rep; 31:2683-2691.
49. Harper DM, et al, GlaxoSmithKline HPV Vaccine Study Group (2004): Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus 16 and 18 in young women: a randomized controlled trial, Lancet; 364:1757-1765.
50. Hawley-Nelson P, et al (1989): The HPV E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes, EMBO J; 8: 3905-3910.
51. Hirose Y, Manley JL (2000): RNA polymerase II and the integration of nuclear events, Genes & Dev; 14: 1415-1429.
52. Hirth JM, et al (2012): Completion of the human papillomavirus vaccine series among insured females between 2006 and 2009, Cancer; 118: 5623-5629.
53. Hirth JM, et al (2013): Completion of the human papillomavirus (HPV) vaccine series among males with private insurance between 2006 and 2009, Vaccine; 31: 1138-1140.
54. Hoque ME, Van Hal G (2014): Acceptability of human papillomavirus vaccine: A survey among master of Business Administration students in KwaZulu Natal, South Africa, Biomed Res Int; Article ID: 257807.
55. Huang Z, Peng S, et al (2015): Combination of proteasome and HDAC inhibitor enhances HPV 16 E-specific CD8 + T cell immune response and antitumor effect in a preclinical cervical cancer model, J Biomed Sci; 22:7.
56. Hwang SW, et al (2001): Human papillomavirus type 16 E7 binds to E2FI and activates E2FI-driven transcription in a retinoblastoma protein-independent manner, J BIol Chem; 227: 2923-2930.
57. Iliya Z, et al (2010): Cervical cancer risk predictor of human papillomavirus acceptance among female university in north Nigeria, J Obstet Gynaecol; 30: 857-862.
58. Jiang P, Yue Y (2014): Human papillomavirus oncoproteins and apoptosis (Review), Exp Ther Med; 7:3-7.
59. Jeon S, et al (1995): Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells, J virol; 69: 2989-2997.
60. Kadaja M, et al (2009): Mechanisms of genomic instability in cells infected with the high-risk human papillomavirus, PLos Pathog; 5: e10003907.
61. Kaspersen MD, Larsen PB, et al (2011): Identification of multiple HPV types in spermatozoa from human sperm donors, PLoS One; 6: e18095.
62. Katich SC, et al (2001): Regulation of cdc 25A gene by the human papillomavirus 16 E7 oncogene, Oncogene; 20: 543-530.
63. Kim MS, Bak Y, et al (2013): Wogonin induces apoptosis by suppressing E6 and E7 expression and activating intrinsic signaling pathway in HPV-16 cervical cancer cells, Cell Biol Toxicol; 29:259-272.
64. Konarska MM, Query CC (2005): Insight into the mechanisms of splicing: more lesson from the ribosome, Genes Dev; 19: 2255-2260.
65. Koskimaa HM, et al (2010): Molecular markers implicating early malignant events in cervical carcinogenesis, Cancer Epidemiology Biomarkers & Prevention; 19: 2003-2012.
66. Larson H (2015): The world must accept that the HPV vaccine is safe, Nature; 3:528.
67. Li Y, et al (2010): High risk human papillomavirus reduces the expression of microRNAs-218 in women with cervical intraepithelial neoplasia, J Int Med Res; 38: 1730-1736.
68. Li Y, et al (2015): A novel dithiocarbamate derivate induces cell apoptosis through p53-dependent intrinsic pathway and suppresses the expression of the E6 oncogene in human papillomavirus 18 in HeLa cells, Apoptosis; 20:787-795.
69. Lin Z, Bazzaro M, et al (2009): Combination of proteasome and HDAC inhibitor for uterine cervical cancer treatment, Clin Cancer Res; 15: 570-577.
70. Liu G, et al (2015): HPV vaccine campaign and dose adherence among commercially insured females aged 9 through 26 years in the US, Papillomavirus Res; 2:1-8.
71. Liu X, et al (2006): Structure of the human papillomavirus E7 oncoprotein: self assemble into defined spherical oligomers, Biochemistry; 43:3310-3317.
72. Longswork MS, Laimins LA (2004): Pathogenesis of human papillomaviruses in differentiating epithelial cells, Microbiol Mol Biol Rev; 68: 362-372.
73. Luckett R, Feldman S (2015): Impact of 2-, 4-, and 9-valent HPV vaccine on morbidity and mortality from cervical cancer, Human Vaccines & Immunotherapeutic; DOI: 10.1080/21645515.2015.1108500.
74. Luhn P, Wentzensen N (2013): HPV-based tests for cervical cancer screening and management of cervical disease, Current Obs & Gynecol Reports; 2:677-700.
75. Lui H, et al (2012): Genomic amplification of the human telomerase gene (hTERC) associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer, Diagnostic Pathol; 7:147
76. Maddry JA, Chen X, et al (2011): Discovery of novel benzoquinazoline and thiazoloimidazoles inhibitors of influenza H5N1 viruses, from a cell-based high-throughput screen, J Biomol Screen; 16: 73-81.
77. Majno G, Joris I (1995): Apoptosis, Oncosis, and Necrosis, Am J Pathol; 146: 3-15.
78. Malta M, et al (2015): Let-7c is a candidate biomarker for cervical cancer intraepithelial lesions: A pilot study, Mol Diagnosis & therapy; 19:191-196.
79. McLanghlin-Drubin ME, Munger K (2009): The human papillomavirus E7 oncoprotein, Virology; 384: 335-344.
80. McQueen F, Duvall E (2006): Using a quality control approach to define an adequately cellular liquid-based cervical cytology specimen, Cytopathology; 17: 168-171.
81. Meijer M, et al (2009): Validation of high-risk HPV testing for primary cervical screening, J of Clinical Virology; 46 (Suppl 3): S1-S4.
82. Mokbel K, Hassanally P (2001): From HER2 to Herceptin, Curr Med Res Opin; 17: 51-59.
83. Moody CA, Laimins LA (2010): Human papillomavirus oncoprotein pathway to transformation, Nature Rev; 10:530-560.
84. Mishra A, Verma M (2010): Cancer biomarkers: Are we ready for the prime time? Cancers; 2: 190-208.
85. Munger DJ, et al (1988): Human papillomavirus type 16 alters human epithelial cell differentiation in vitro, PNAS USA; 85: 7169-7173.
86. NCI : http://www.cancer.gov/publications/dictionaries/cancer-terms/?CdrID=45618 (Accessed on December 14, 2015)
87. Nichols AC, Palma DA, et al (2013): High frequency of activating PIK3CA mutation in human papillomavirus-positive orpharyngeal cancer, JAMA Otolaryngol Head Neck Surg; 139: 617-622.
88. Nyugen CL, Munger K (2008): Direct association of the HPV 16 E7 oncoprotein with cyclin A/CDK2 and cyclin E/CDK2 complexes, Virology; 380: 21-25.
89. Nyugen DX, et al (2002): Human papillomavirus type 16 E7 maintained elevated levels of the cdc 25A tyrosine phosphatase during deregulation of cell cycle arrest, J Virol; 76: 619-632.
90. Patel DA, Patel AC, et al (2012): High-throughput for small molecules enhancers of the interferon signaling pathway to drive next-generation antiviral drug discovery, PLoS One; 7: e36594.
91. Patel DA, Patel AC, et al (2014): High-throughput screening normalized to biological response: application to antiviral drug discovery, J Biomed Screen; 19.10.117711087957113496848.
92. Patrick DR, et al (1994): Identification of a novel retinoblastoma gene product binding site in human papillomavirus type 16 E7 protein, J Biol Chem; 269: 6842-6850.
93. Peng S, et al (2004): Development of a DNA vaccine targeting human papillomavirus type 16 oncoproteins E6, J Virol; 78: 8468-8476.
94. Phelps WC, et al (1988): The human papillomavirus type 16 E7 gene encodes transactivation and transformation function similar to those of adenovirus E1A, Cell; 53: 539-547.
95. Pisani P, Bray F, Parkin DM (2002): Estimates of the world-wide prevalence of cancer for 25 sites in the adult population, Int J Cancer; 97:72-81.
96. Puig-Basagoiti F, Deas TS, et al (2005): High-throughput assays using a luciferase expressing Replicon, virus-like particles, and full length virus for West Nile virus drug discovery, Antomicrob Agents Chemother; 49: 4980-4988.
97. Rasmussen L, Maddox C, et al (2011): A high-throughput screening strategy to overcome virus instability, Assay Drug Dev Technol; 9: 184-190.
98. Rauber D, et al (2008): Prognostic significance of the detection of human papillomavirus L1 protein in smears of mild to moderate cervical intraepithelial lesions, Eur J Obs & Gynecol; 140: 258-260
99. Ressing ME, Sette A, et al (1995): Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides, J Immunol; 154:5934-5943.
100. Ribeiro J, et al (2015): miR-34a and miR-125b expression in HPV infection and cervical cancer development, Biomed Res Int; 2015:304584.
101. Ronco G, et al (2010): Efficacy of human papillomavirus testing for the detection of invasive cervical cancer and cervical intraepithelial neoplasia: a randomized controlled study, The Lancet Oncol; 11: 249-257.
102. Saha B, et al (2007): Telomerase and markers of cellular proliferation are associated with the progression of cervical intraepithelial neoplasia lesions, Int J Gynecol; 26: 214-222.
103. SBIR-STRR: Apovax104- HPV as a novel vaccine for cervical cancer on https://www.sbir.gov/sbirsearch/detail/91754 (Accessed on 13th December,2015).
104. Schiffman M, et al (2005): The carcinogenicity of human papillomaviruses types reflects viral evolution, Virology; 357: 76-84.
105. Schiker JT, et al (2008): An update of prophylactic human papillomavirus L1 virus-like particle vaccine clinical trials results, Vaccine; 26: 53-61.
106. Schlecht NF, Kulagu S, et al (2001): Persistent human papillomavirus as a predictor of cervical cancer intraepithelial neoplasm, HAMA; 286: 3406-3114.
107. Schwarz E, et al (1985): Structure and transcription of human papillomavirus sequences in cervical carcinoma cells, Nature; 314:111-114.
108. Shi Y, et al (2002): Enhanced sensitivity of multiple myeloma cells containing PTEN mutations to CCGI-779, Cancer Res; 62: 5027-5034.
109. Smith DJ, et al (2008): “Nought may endure but mutability”: spliceosome dynamics and the regulation of splicing, Mol Cell; 30: 657-666.
110. Smith PP, et al (1992): Viral integration and fragile sites in human papillomavirus-immortalized human keratinocytes cell lines, Genes Chromosome Cancer; 5:152-157.
111. Stockwell BR, et al (1999): High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications, Chem Biol; 6: 71-83.
112. Stockwell BR (2004): The biological magic behind the bullets, Nat Biotechnology; 22: 37-38.
113. Stoilov P, et al (2008): A high-throughput screening strategy identifies a cardotomic steroids as alternative splicing modulators, PNAS USA; 105: 11218-11223.
114. Stubenrauch F, et al (1998): Differentiation requirement for conserved E2 binding sites in the life cycle of oncogenic human papillomavirus type 31, J virol; 72: 1071-1077.
115. Tewari KS, et al (2014): Improved survival with bevacizumab in advanced cervical cancer, NEJM; 370:734-743.
116. The Biomarkers Consortium on www.biomarkersconsortium.org (Accessed on December 14, 2015)
117. Thierry F, Yaniv M (1987): The BOV1-E2 trans-acting protein can be either an activator or a repressor of the HPV-18 regulatory region, EMBO J; 6:3391-3397.
118. Tornesello ML, et al (2011); Viral and cellular biomarkers in the diagnosis of cervical intraepithelial neoplasia and cancer, Biomed Res Int; Article ID: 519619.
119. Van Doorslaer K, Burk RD (2012): Association between hTERT activation as HPV E6 protein and oncogenic risk, Virology; 433:216-219.
120. Venuti A, Paolini F, et al (2011): Papillomavirus E5: the smallest oncoprotein with many functions, Mol Cancer; 10:40.
121. Villa LL, et al (2005): Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomized double-blind placebo-controlled multicenter phase II efficacy trial, Lancet Oncol; 6:271-278.
122. Wahl MC, et al (2009): The spliceosome: design principles of a dynamic RNP machine, Cell; 136: 701-718.
123. Walboomen JM, et al (1999): Human papillomavirus is a necessary cause of invasive cervical cancer worldwide, J Pathol; 189: 12-19.
124. Wang H, Yu J, Li L (2015): A DNA vaccine encoding mutate HPV 58mE6E7-Fc-GPI fusion antigen and GM-CSF and B7.1, Oncogene; 8: 3062-3077.
125. Wieringa HW, et al (2015): Breaking the DNA damage response to improve cervical cancer treatment, Cancer Treat Rev; pii: S0305-7372 [Epub ahead of print].
126. Xiao H, et al (2014): Expression of Yes-associated protein in cervical carcinoma epithelial lesions, Int J Gynecol Cancer; 24: 1575-1582.
127. Xu J, et al (2012): Expressed miR-24 expression via Upregulation of target gene chk1 contributes to the progression of cervical cancer, Oncogene; 32: 976-987.
128. Yuan CH, Filippova M, Duerksen-Hughes P (2012): Modulation of apoptotic pathways by human papillomavirus (HPV): mechanisms and implication for therapy, Viruses; 4: 3831-3850.
129. Zang R, Li D, et al (2012): Cell-based assays in high-throughput screening discovery, Int J Biotech for Wellness industries; 1: 31-51.
130. Zerfass-Thomas K, et al (1996): Inactivation f the cdk inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein, Oncogene; 13: 2323-2330.
131. Zimet GD, Rosenthal SL (2010): HPV vaccine among males: issues and Challenges, Gynecol Oncol; 117: (2 Suppl 1): S26-31.
132. zur Hausen H (2002): Papillomavirus and cancer: from basic studies to clinical application, Nat Rev; 2: 342-350.
133. Zur Hausen H (2009): Papillomavirus in the causation of human cancers: a brief historical account, Virology; 384: 260-265.