**4. Molecular therapies in development**

cisplatin, and so non-platinum chemotherapy or higher doses of cisplatin, in these cases, are indicated [81, 91]. In the cases of cisplatin resistance or disease recurrence, non-platinum-based agents such as topotecan, vinorelbine, irinotecan, paclitaxel, mitomycin c, and ifosfamide are sometimes combined with cisplatin. Topotecan and 5-fluorouracil (5-FU), among other combinations, seem to produce an additive effect with cisplatin to reduce its toxicity, increasing its RR from 20 to 50 percent [90, 91, 95]. Similarly, when paclitaxel is combined with cisplatin, a high RR of 46 percent is reached for late stage IV cervical cancer and is accompanied by decreased hematologic complications. However, a Gynecologic Oncologic group study reported that consistent, weekly schedules of cisplatin alone are less toxic than cisplatin combined with other agents, particularly 5-FU [92, 96]. Sanazol and tirapazamine are relatively new chemotherapeutic agents that specifically target and destroy hypoxic tissue by dissociat‐ ing into free radicals that cause DNA damage. Therefore, drug selectivity for hypoxic tissue will result in greater cytotoxicity among malignant cervical cells [81]. Multiple-agent regimens may also include the use of antibodies targeting a tumor's peculiar characteristics. For example, if a particular tumor markedly over-expresses EGFR-1, it would be appropriate to include

298 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Cetuximab in treatment, or Bevacizumab in the case of extreme vascularity [95].

study combination therapies.

*Combination Therapies:* Multidisciplinary treatment might be indicated throughout any of the stages of cervical carcinoma, mainly depending on its aggressiveness. In fact, it is quite common for treatment schedules to include chemotherapy, radiation therapy, and surgery [81]. The concurrent use of chemotherapy and radiation therapy is reported by the NCI to reduce cervical cancer mortality by 30 to 50 percent, particularly in late stage II. Alternatively, neoadjuvant therapy, defined as a specific sequence for delivering any treatment before a definitive therapy such as surgery or radiotherapy, may be employed. Neoadjuvant therapy is intended to prime the target tissue, thus making it more susceptible to primary treatment [71, 93]. Neoadjuvant chemotherapy (NACT) is often administered before radiation in order to radiosensitize solid tumor cells and to decrease tumor size and hypoxic cell numbers. In few instances, NACT could potentially provide patients with the option of surgery even though it may have been unfeasible prior to NACT. Moreover, researchers are finding that patients who receive sequential NACT-RH have a 10 to 15 percent survival advantage five years after treatment [97]. In cases when surgery does not completely remove all traces of abnormal tissue as anticipated, chemotherapy or radiation must be given post-operatively to inhibit local and distal metastasis through the lymphatic system. Hence, there is no doubt that concurrent chemotherapy and radiation therapy can improve survival in women with locally advanced cervical cancer or recurrent cancer [93, 98]. Radiation treatment alone does not contain cancer in 35 to 90 percent of patients, but chemotherapy given with radiation treatment yields much higher survival rates. The chemotherapeutic drugs most commonly used with radiation are cisplatin, 5-FU, mitomycin C, and hydroxyurea, though cisplatin produces the largest increase in survival by reducing mortality and recurrence [93, 94]. Many times, the sensitizing effects of drugs are needed to accentuate the value of other treatment methods, as is the case with histone deacetylase inhibitors, decitabine and valproic acid, that radiosensitize tumors for RT [81]. Thus, researchers may build and forge new applications through trials that

*Therapeutic Immune Strategies:* The development of the prophylactic vaccine has forever changed the course of HPV-mediated cervical disease. Nevertheless, it is clear that there is still an immense need for therapeutic options, especially in developing countries where the positive, yet costly measures of preventative initiatives remain to be implemented. In contrast to prophylactic vaccines that target the L1 and L2 proteins and are protective against HPV infections, therapeutic vaccines would ideally target molecules such as E6 and/or E7 postinfection, which are directly linked to HPV-mediated carcinogenesis [99]. Therapeutic vaccines may be constructed in a variety of ways, as described below [100].

Live, vector-based vaccines, bacterial and viral, can generate very robust cell-mediated and adaptive immune responses, and because of this they are preferred over peptide/protein vaccines. Specifically, bacterial vectors function well when they are packaged with antigen (genes or proteins), thereby alerting antigen-presenting cells (APCs) to initiate an immune response. Though several bacterial vectors have been tested, *L. monocytogenes* is a prototypic example. Simply, *L. monocytogenes* stimulates antigen-specific CD8+ and CD4+ T cell responses following its evasion of immune destruction by releasing *Lm* toxin to avoid phagosomal lysis. However, the most appealing factor of the *L. monocytogenes* vector is that the immune response can be easily controlled by antibiotics should the body react adversely to *Lm* [101-104]. With regards to viral vectors, a few viruses, such as the vaccinia virus, adenovirus, vesicular stomatitis virus, and alphavirus, have distinguished themselves and show great promise. In fact, researchers have discovered that when an adenovirus vector is used to deliver calreticulin and HPV E7 antigens, the size of E7-expressing tumors in mice decrease [105]. A highly anticipated viral vaccine candidate is the TA-HPV vaccine, consisting of both HPV16 and HPV18 E6 and E7 antigens and a vaccinia virus vector. TA-HPV is safe and efficient in stimulating either a specific CTL response or a serological response, which might depend on the epigenetic patterns of each individual [106]. Similarly, the MVA E2 vaccine is also packaged with the vaccinia virus, and uses the bovine papillomavirus E2 protein to repress E6 and E7 transcription. MVA-HPV-IL2, currently undergoing a phase III clinical trial for CIN 2-3 treatment, utilizes a modified vaccinia Ankara viral vector, and uniquely contains HPV16 E6 and E7 DNA as well as IL-2 [99, 107]. The co-expression of a cytokine with HPV antigens induces a stronger immune response by stimulating dendritic cell maturation, though the refinement of viral vector tools must include solutions for overcoming pre-existing immunity. To rectify this, Cox-2 inhibitors are presently being tested to offset such immune interferences, thus allowing greater exploitation of a potentially powerful treatment. However, safety factors remain a high priority when viral vectors are considered, and these vectors must be properly constructed for use in both immunocompetent and immunocompromised individuals [100].

In peptide-based vaccines, antigens from HPV are directly administered to elicit a response from dendritic cells (DCs) *via* toll-like receptor (TLR) activation [108]. The peptide vaccine platform is ideal for mass production, but the breadth of its efficacy is limited by the expression of only one major histocompatibility complex I (MHC I) phenotype; protein-based vaccines are not encumbered in the same way. However, if the specific immunogenic epitopes on peptides could be identified it would greatly remedy this difficulty. Some investigators are taking a different approach by overlapping peptides with a broad range of different epitopes to obtain a greater immune response [109]. Meanwhile, other researchers have focused on the development of vaccines that utilize a synthetic E7 peptide component to clear HPV-mediated tumors in mice, such as TriVax [110]. Unfortunately, a limitation of both peptide- and proteinbased vaccines is low immunogenicity. This challenge, however, is no longer intractable with the advent of various immunomodulatory adjuvant agents such as TLR ligands, cytokines, and lipids, all of which help to stimulate a robust immune response. Another recently popular strategy thought to increase protein/peptide vaccine potency involves the Pan HLA-DR epitope peptide (PADRE), which binds MHC class II molecules with much stronger affinity [108]. Following the success of PADRE and other similar technologies, more potent enhancers of peptide vaccines such as 4-1BB ligand, CpG oligodeoxynucleotide, mutant cholera toxin, and lipopeptides are now emerging [111].

focus on antigen modifications so as to elicit a stronger DC adaptive immune response. One such strategy increases the number of HPV DNA plasmid transfection events in DCs. These DCs will then present antigen to, and ultimately activate, naive CD4+ and CD8+ lymphocytes [119]. However, researchers still must determine the most efficient and effective way to deliver HPV DNA to DCs. A fairly recent investigation discusses a novel method to administer a dosedriven vaccine by gene gun technology, which forms a DNA-coated stream of gold particles targeting Langerhans cells in the skin [120]. Other studies justify the use of cell membrane permeabilization by electroporation, thereby causing cells to experience an electric shock and

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301

Electroporation also leads to inflammation and cytokine recruitment, thus enhancing the immune environment. Additionally, electroporation was found to be particularly effective against E7-expressing tumors. The VGX-3100 plasmid DNA vaccine, targeting E6 and E7 antigens of HPV16 and 18, seemed to have great efficacy when it was combined with electro‐ poration administration [123]. Furthermore, clinical trials attest to the value of this particular method of treatment delivery in CIN 2 and 3 lesions. Strategies that increase transfection efficiency are continuously being sought through experimentation with diverse routes of vaccine administration, such as intramuscular *versus* intradermal techniques. The efficient intramuscular administration of DNA is achieved *via* microencapsulation, which uses a biopolymer that surrounds the plasmid to prevent degradation by nucleases. Conversely, intradermal administration involves skin patch tattooing using microneedles [124]. One encapsulated DNA-based vaccine that has been tested with both administrative routes is the amolimogene bepiplasmid (also known as ZYC101a), which contains T cell epitopes and HPV16 and 18 E6/E7 viral protein fragments [125, 126]. In this study, it was concluded that

Another strategy to strengthen DNA-based vaccines focuses on improving DC antigen processing. Those cells that have become transfected with HPV DNA material may be prompted to generate a more potent immune response through codon optimization or demethylation techniques that will increase gene translation efficiency [100]. These methods work to improve antigen translation and expression in cells with HPV DNA. Additionally, DNA vaccination with the MHC class I chaperone molecule, calreticulin, was shown to increase the CD8+ immune response, thereby leading to an antitumor effect [129]. It is also possible to improve antigen processing through the MHC class II pathway. For instance, the E7/LAMP-1 vaccine allows antigen to be further sorted in endosomal and lysosomal compart‐ ments, thus priming CD4+ and CD8+ lymphocytes for a greater response as compared to the administration of E7 alone [130]. Substitution of the MHC class II peptide, CLIP (Class IIassociated peptide), for the PADRE peptide in the invariant chain is a promising strategy to not only increase antigen presentation, but also to secrete cytokines that stimulate T cell proliferation, thus resulting in greater CD4+ lymphocyte activity [131, 132]. Other methods of improving antigen presentation include cross-presentation by extracellular proteins like HSP 70, up-regulation of MHC II expression on the surface of DCs, and single chain trimer technology (SCT). SCT involves the fusion of HPV antigen to the MHC class I molecule, beta-2 microglobulin, resulting in the appropriate recognition of antigen and action against an E6-

maximizing cellular uptake of DNA [121, 122].

intramuscular methods were more effective [127, 128].

expressing tumor [133, 134].

In general, protein-based therapeutic vaccines, like peptide-based vaccines, are advantageous for safety and tolerability. Although protein-based vaccines are not restricted by MHC compatibility, they cannot directly stimulate cytotoxic T lymphocytes. Protein vaccine adjuvants that are considered to compensate for this weakness in protein vector therapy include liposome-polycation-DNA and the saponin-based ISCOMATRIX. The ISCOMATRIX is an adjuvant complex consisting of phospholipids and cholesterols, and it causes a rapid innate immune cell response [112]. In general, any strategy that increases antigen uptake by APCs, antigen presentation, or the CTL response is expected to improve the immunogenicity of a protein. One protein-based therapeutic vaccine in clinical trials is TA-CIN. Essentially, TA-CIN is a mixture of L2, E6, and E7 proteins from HPV16. The L2 antigen launches a humoral response, and the E6 and E7 proteins induce T cell responses. However, further investigation revealed that TA-CIN is even more powerful when combined with the TA-HPV vaccine [113-115]. Another strong protein-based vaccine candidate, due to its safety and ability to induce lesion regression in various HPV-related diseases, is HspE7 [116]. HspE7 is a fusion product of HPV16 E7 and the *Mycobacterium bovis* Hsp65 proteins. Another potential strategy to improve immunogenicity in protein-based therapeutic vaccines is the use of the Fve adjuvant, which is derived from a fungal protein originating in the *Flammulina velutipes* species, and has been shown to produce potent humoral and cellular immune responses. The antitumor effects of Fve in HPV-mediated cancers are attributed to its ability to induce IFN-gamma secretion and to stimulate T helper and CTLs in tumor-bearing mice [117]. Our knowledge about how to apply protein and peptide therapeutic vaccines against cervical cancer is steadily increasing. The immediate next step is to follow up with successful clinical trials, and to implement the most useful of these methods, or a combination thereof.

One advantage of DNA-based vaccination is its capacity to increase immunological memory through constant antigen production. Because the immune response itself is not anti-vector, multiple vaccinations are possible. Moreover, the antigens produced by DNA vaccines can be delivered in a variety of ways, resulting in stimulation of both APCs and T lymphocyte immune defenses [118, 119]. However, DNA vaccines also present the challenge of overcoming low immunogenicity due to limited APC specificity. Therefore, future developments must focus on antigen modifications so as to elicit a stronger DC adaptive immune response. One such strategy increases the number of HPV DNA plasmid transfection events in DCs. These DCs will then present antigen to, and ultimately activate, naive CD4+ and CD8+ lymphocytes [119]. However, researchers still must determine the most efficient and effective way to deliver HPV DNA to DCs. A fairly recent investigation discusses a novel method to administer a dosedriven vaccine by gene gun technology, which forms a DNA-coated stream of gold particles targeting Langerhans cells in the skin [120]. Other studies justify the use of cell membrane permeabilization by electroporation, thereby causing cells to experience an electric shock and maximizing cellular uptake of DNA [121, 122].

peptides could be identified it would greatly remedy this difficulty. Some investigators are taking a different approach by overlapping peptides with a broad range of different epitopes to obtain a greater immune response [109]. Meanwhile, other researchers have focused on the development of vaccines that utilize a synthetic E7 peptide component to clear HPV-mediated tumors in mice, such as TriVax [110]. Unfortunately, a limitation of both peptide- and proteinbased vaccines is low immunogenicity. This challenge, however, is no longer intractable with the advent of various immunomodulatory adjuvant agents such as TLR ligands, cytokines, and lipids, all of which help to stimulate a robust immune response. Another recently popular strategy thought to increase protein/peptide vaccine potency involves the Pan HLA-DR epitope peptide (PADRE), which binds MHC class II molecules with much stronger affinity [108]. Following the success of PADRE and other similar technologies, more potent enhancers of peptide vaccines such as 4-1BB ligand, CpG oligodeoxynucleotide, mutant cholera toxin,

300 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

In general, protein-based therapeutic vaccines, like peptide-based vaccines, are advantageous for safety and tolerability. Although protein-based vaccines are not restricted by MHC compatibility, they cannot directly stimulate cytotoxic T lymphocytes. Protein vaccine adjuvants that are considered to compensate for this weakness in protein vector therapy include liposome-polycation-DNA and the saponin-based ISCOMATRIX. The ISCOMATRIX is an adjuvant complex consisting of phospholipids and cholesterols, and it causes a rapid innate immune cell response [112]. In general, any strategy that increases antigen uptake by APCs, antigen presentation, or the CTL response is expected to improve the immunogenicity of a protein. One protein-based therapeutic vaccine in clinical trials is TA-CIN. Essentially, TA-CIN is a mixture of L2, E6, and E7 proteins from HPV16. The L2 antigen launches a humoral response, and the E6 and E7 proteins induce T cell responses. However, further investigation revealed that TA-CIN is even more powerful when combined with the TA-HPV vaccine [113-115]. Another strong protein-based vaccine candidate, due to its safety and ability to induce lesion regression in various HPV-related diseases, is HspE7 [116]. HspE7 is a fusion product of HPV16 E7 and the *Mycobacterium bovis* Hsp65 proteins. Another potential strategy to improve immunogenicity in protein-based therapeutic vaccines is the use of the Fve adjuvant, which is derived from a fungal protein originating in the *Flammulina velutipes* species, and has been shown to produce potent humoral and cellular immune responses. The antitumor effects of Fve in HPV-mediated cancers are attributed to its ability to induce IFN-gamma secretion and to stimulate T helper and CTLs in tumor-bearing mice [117]. Our knowledge about how to apply protein and peptide therapeutic vaccines against cervical cancer is steadily increasing. The immediate next step is to follow up with successful clinical trials, and to

implement the most useful of these methods, or a combination thereof.

One advantage of DNA-based vaccination is its capacity to increase immunological memory through constant antigen production. Because the immune response itself is not anti-vector, multiple vaccinations are possible. Moreover, the antigens produced by DNA vaccines can be delivered in a variety of ways, resulting in stimulation of both APCs and T lymphocyte immune defenses [118, 119]. However, DNA vaccines also present the challenge of overcoming low immunogenicity due to limited APC specificity. Therefore, future developments must

and lipopeptides are now emerging [111].

Electroporation also leads to inflammation and cytokine recruitment, thus enhancing the immune environment. Additionally, electroporation was found to be particularly effective against E7-expressing tumors. The VGX-3100 plasmid DNA vaccine, targeting E6 and E7 antigens of HPV16 and 18, seemed to have great efficacy when it was combined with electro‐ poration administration [123]. Furthermore, clinical trials attest to the value of this particular method of treatment delivery in CIN 2 and 3 lesions. Strategies that increase transfection efficiency are continuously being sought through experimentation with diverse routes of vaccine administration, such as intramuscular *versus* intradermal techniques. The efficient intramuscular administration of DNA is achieved *via* microencapsulation, which uses a biopolymer that surrounds the plasmid to prevent degradation by nucleases. Conversely, intradermal administration involves skin patch tattooing using microneedles [124]. One encapsulated DNA-based vaccine that has been tested with both administrative routes is the amolimogene bepiplasmid (also known as ZYC101a), which contains T cell epitopes and HPV16 and 18 E6/E7 viral protein fragments [125, 126]. In this study, it was concluded that intramuscular methods were more effective [127, 128].

Another strategy to strengthen DNA-based vaccines focuses on improving DC antigen processing. Those cells that have become transfected with HPV DNA material may be prompted to generate a more potent immune response through codon optimization or demethylation techniques that will increase gene translation efficiency [100]. These methods work to improve antigen translation and expression in cells with HPV DNA. Additionally, DNA vaccination with the MHC class I chaperone molecule, calreticulin, was shown to increase the CD8+ immune response, thereby leading to an antitumor effect [129]. It is also possible to improve antigen processing through the MHC class II pathway. For instance, the E7/LAMP-1 vaccine allows antigen to be further sorted in endosomal and lysosomal compart‐ ments, thus priming CD4+ and CD8+ lymphocytes for a greater response as compared to the administration of E7 alone [130]. Substitution of the MHC class II peptide, CLIP (Class IIassociated peptide), for the PADRE peptide in the invariant chain is a promising strategy to not only increase antigen presentation, but also to secrete cytokines that stimulate T cell proliferation, thus resulting in greater CD4+ lymphocyte activity [131, 132]. Other methods of improving antigen presentation include cross-presentation by extracellular proteins like HSP 70, up-regulation of MHC II expression on the surface of DCs, and single chain trimer technology (SCT). SCT involves the fusion of HPV antigen to the MHC class I molecule, beta-2 microglobulin, resulting in the appropriate recognition of antigen and action against an E6 expressing tumor [133, 134].

RNA replicon-based vaccines have some advantages over DNA vaccines: 1) they are less likely to integrate into the host genome, thus decreasing the risk of cell transformation and 2) they can potentially generate more protein than can DNA methods. Of course, RNA replicon-based vaccines may be introduced into the host as DNA. From here, the cell can then transcribe the DNA molecule into RNA, but without the structural genes needed to construct viral particles. Therefore, no antibodies are produced against viral immunologic molecules and administra‐ tion can be repeated. One significant limitation of using replicons is that RNA is inherently unstable. However, the use of a DNA-launched RNA replicon could surmount this difficulty, and concerns of gene integration could be addressed by designing the DNA to self-destruct following gene expression. Because immunologic cells undergo apoptosis in this process, it is necessary to fuse the HPV antigen to an anti-apoptotic protein, otherwise DC numbers will be drastically reduced [100, 135, 136]. The Kunjin flavivirus has the potential to accomplish the same goal by delivering the desired antigens into cells without immediately inducing apop‐ tosis, thus prolonging the window of time for antigen presentation by transfected cells and improving overall immunogenicity [137-139].

Of course, every approach has its advantages and disadvantages, but combining several therapeutic vaccines into a single regimen may offer synergy and thus strengthen treatment efficacy. For example, one preclinical study tested a prime-boost vaccination model. The immune system was first primed with a DNA vaccine consisting of HPV16 E7 and LAMP-1 (Sig/E7/LAMP-1). Then, a booster dose of Sig/E7/LAMP-1 was given again to maintain and increase the T-cell response over a longer period [150]. Because several prime-boost studies have yielded continuous positive results in safety and efficacy, we can expect to see similar

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*RNA-based Therapy:* RNAs have the unique ability to form double-stranded molecules by hybridizing with complementary antisense RNA. It was established years ago that antisense E6/E7 RNAs in a plasmid could stagnate cellular growth [151]. More recently, the ability to design antisense oligodeoxynucleotides (ODNs) that specifically bind E6/E7 RNA molecules at the translation initiation region with high affinity have made the use of translation inhibition more feasible for achieving cervical carcinoma treatment goals. Later studies have confirmed these results, and suggest that stronger, additive effects may also occur when antisense ODNs

Tristetrapolin (TTP) is an RNA-binding protein with anti-cancer properties, and yields its effects by binding to AU rich regions of mRNA and promoting their destruction. These AUrich elements (AREs) of mRNA, located in the untranslated region of the strand, are naturally involved in regulating cellular growth and inflammation *via* mediators such as TNF-alpha and COX-2. Therefore, the affinity with which TTP binds these elements makes this interaction an

higher levels of p53 as compared to untreated cells. Additionally, in the presence of TTP, these same cells acquired the ability to inhibit E6-AP expression, suggesting a possible mechanism for the rescue of p53 [154]. Other RNA-based methods targeting HPV include the use of siRNAs directed against E6 and E7. siRNAs cause the cleavage and degradation of homologous sequences through their participation in the RNA-induced Silencing Complex (RISC). Re‐ searchers are now investigating the use of vehicles such as shRNAs to target and destroy RNAs of interest. However, better systems for delivering shRNAs to the nuclei, and better ways to

*Antibody-based Therapy:* The use of monoclonal antibodies in cancer treatment is an appealing concept due to the selectivity and specificity with which an antibody can bind to the molecule of interest. Molecules participating in tumor progression can be targeted by antibodies through three general mechanisms: 1) Recognition of specific tumor-associated receptors, such as EGFR; 2) Binding to immune effector cells, and 3) Binding to tumor-promoting molecules such as VEGF. Though no monoclonal antibodies have been approved for the treatment of cervical cancer, researchers are accruing more convincing evidence of their value [156]. As with most cancers, cervical carcinomas possess a dynamic vascular network. Thus, much investigation has gone into developing biologic agents that target molecular pathways associated with vascularization, such as those involving vascular endothelial growth factor (VEGF). Bevaci‐ zumab is an angiogenesis inhibitor that ultimately delays vasculogenic processes, and has long been used effectively in the treatment of other malignancies such as colorectal cancer. The

HeLa cells exposed to TTP demonstrated

combinatory therapeutic trials in the future.

are designed with adjacent mRNA targets in mind [152, 153].

access cellular uptake mechanisms for siRNAs, are needed [155].

attractive point of intervention. For example, HPV+

Dendritic cell-based vaccines can be prepared in several ways: by introducing exogenous HPV antigen via endocytosis in to DCs; by infusing DCs with E6/E7 DNA or RNA through electropo‐ ration; or, the antigen may be packaged together with liposomes or nanoparticles to be deliv‐ eredintoDCs [140].DCinteractionswithTcellsandthe subsequentperpetuationofthe immune signal are essential features that determine whether an organism will demonstrate a strong immune response, or whether it will exhibit immune tolerance (e.g. if the DCs are immature) [141, 142]. Essentially, DCs activate T cells and T cells, in turn, mediate DC apoptosis. There‐ fore, it has been proposed that prolonging DC survival may strengthen and lengthen the initial T cell stimulation [143]. However, because the idea of combining HPV vaccines with antiapoptotic proteins has not gained much popularity due to the possibility of cellular transforma‐ tion, other approaches such as co-administering vaccines with siRNAs targeting proapoptotic proteins are gaining traction. Designing shRNAs directed at FasL produced by DCs to promote T cell apoptosis, for example, could increase the number of T cells stimulated [144]. DCactivationcanalsobeprolongedbydeactivatingthenegativeregulationofcytokinesignaling through SOCS-1, which acts on the Jak-Stat pathway [132, 145, 146].

Because tumor cell-based vaccines have shown promise in malignancies like melanoma, colon and prostate cancers, many subscribe to this paradigm as the key to solving the cervical carcinoma dilemma. The idea of manipulating tumor cells into becoming more discernible by the immune system is based on their expression of immunomodulatory cytokines like IL-2 and IL-12 [147]. Other studies have found that engineering tumor cells to secrete pro-immune cytokines such as GM-CSF produces antitumor immunity as well [148]. The advantage of using tumor cell-based vaccines is that multiple antigens can be targeted on the surface of a tumor, thus increasing the chance that a single cell or group of cells expressing those antigens will be eliminated by the immune system. As can be expected, such an individualized treatment is costly and may border on the impractical as compared to other recent advances in the field of cervical cancer vaccination. Furthermore, patients who qualify for tumor cell-based vaccina‐ tion would be at greater risk in the receipt of new cancer cells than if they were to employ a treatment plan composed of existing therapies [149].

Of course, every approach has its advantages and disadvantages, but combining several therapeutic vaccines into a single regimen may offer synergy and thus strengthen treatment efficacy. For example, one preclinical study tested a prime-boost vaccination model. The immune system was first primed with a DNA vaccine consisting of HPV16 E7 and LAMP-1 (Sig/E7/LAMP-1). Then, a booster dose of Sig/E7/LAMP-1 was given again to maintain and increase the T-cell response over a longer period [150]. Because several prime-boost studies have yielded continuous positive results in safety and efficacy, we can expect to see similar combinatory therapeutic trials in the future.

RNA replicon-based vaccines have some advantages over DNA vaccines: 1) they are less likely to integrate into the host genome, thus decreasing the risk of cell transformation and 2) they can potentially generate more protein than can DNA methods. Of course, RNA replicon-based vaccines may be introduced into the host as DNA. From here, the cell can then transcribe the DNA molecule into RNA, but without the structural genes needed to construct viral particles. Therefore, no antibodies are produced against viral immunologic molecules and administra‐ tion can be repeated. One significant limitation of using replicons is that RNA is inherently unstable. However, the use of a DNA-launched RNA replicon could surmount this difficulty, and concerns of gene integration could be addressed by designing the DNA to self-destruct following gene expression. Because immunologic cells undergo apoptosis in this process, it is necessary to fuse the HPV antigen to an anti-apoptotic protein, otherwise DC numbers will be drastically reduced [100, 135, 136]. The Kunjin flavivirus has the potential to accomplish the same goal by delivering the desired antigens into cells without immediately inducing apop‐ tosis, thus prolonging the window of time for antigen presentation by transfected cells and

302 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Dendritic cell-based vaccines can be prepared in several ways: by introducing exogenous HPV antigen via endocytosis in to DCs; by infusing DCs with E6/E7 DNA or RNA through electropo‐ ration; or, the antigen may be packaged together with liposomes or nanoparticles to be deliv‐ eredintoDCs [140].DCinteractionswithTcellsandthe subsequentperpetuationofthe immune signal are essential features that determine whether an organism will demonstrate a strong immune response, or whether it will exhibit immune tolerance (e.g. if the DCs are immature) [141, 142]. Essentially, DCs activate T cells and T cells, in turn, mediate DC apoptosis. There‐ fore, it has been proposed that prolonging DC survival may strengthen and lengthen the initial T cell stimulation [143]. However, because the idea of combining HPV vaccines with antiapoptotic proteins has not gained much popularity due to the possibility of cellular transforma‐ tion, other approaches such as co-administering vaccines with siRNAs targeting proapoptotic proteins are gaining traction. Designing shRNAs directed at FasL produced by DCs to promote T cell apoptosis, for example, could increase the number of T cells stimulated [144]. DCactivationcanalsobeprolongedbydeactivatingthenegativeregulationofcytokinesignaling

Because tumor cell-based vaccines have shown promise in malignancies like melanoma, colon and prostate cancers, many subscribe to this paradigm as the key to solving the cervical carcinoma dilemma. The idea of manipulating tumor cells into becoming more discernible by the immune system is based on their expression of immunomodulatory cytokines like IL-2 and IL-12 [147]. Other studies have found that engineering tumor cells to secrete pro-immune cytokines such as GM-CSF produces antitumor immunity as well [148]. The advantage of using tumor cell-based vaccines is that multiple antigens can be targeted on the surface of a tumor, thus increasing the chance that a single cell or group of cells expressing those antigens will be eliminated by the immune system. As can be expected, such an individualized treatment is costly and may border on the impractical as compared to other recent advances in the field of cervical cancer vaccination. Furthermore, patients who qualify for tumor cell-based vaccina‐ tion would be at greater risk in the receipt of new cancer cells than if they were to employ a

improving overall immunogenicity [137-139].

through SOCS-1, which acts on the Jak-Stat pathway [132, 145, 146].

treatment plan composed of existing therapies [149].

*RNA-based Therapy:* RNAs have the unique ability to form double-stranded molecules by hybridizing with complementary antisense RNA. It was established years ago that antisense E6/E7 RNAs in a plasmid could stagnate cellular growth [151]. More recently, the ability to design antisense oligodeoxynucleotides (ODNs) that specifically bind E6/E7 RNA molecules at the translation initiation region with high affinity have made the use of translation inhibition more feasible for achieving cervical carcinoma treatment goals. Later studies have confirmed these results, and suggest that stronger, additive effects may also occur when antisense ODNs are designed with adjacent mRNA targets in mind [152, 153].

Tristetrapolin (TTP) is an RNA-binding protein with anti-cancer properties, and yields its effects by binding to AU rich regions of mRNA and promoting their destruction. These AUrich elements (AREs) of mRNA, located in the untranslated region of the strand, are naturally involved in regulating cellular growth and inflammation *via* mediators such as TNF-alpha and COX-2. Therefore, the affinity with which TTP binds these elements makes this interaction an attractive point of intervention. For example, HPV+ HeLa cells exposed to TTP demonstrated higher levels of p53 as compared to untreated cells. Additionally, in the presence of TTP, these same cells acquired the ability to inhibit E6-AP expression, suggesting a possible mechanism for the rescue of p53 [154]. Other RNA-based methods targeting HPV include the use of siRNAs directed against E6 and E7. siRNAs cause the cleavage and degradation of homologous sequences through their participation in the RNA-induced Silencing Complex (RISC). Re‐ searchers are now investigating the use of vehicles such as shRNAs to target and destroy RNAs of interest. However, better systems for delivering shRNAs to the nuclei, and better ways to access cellular uptake mechanisms for siRNAs, are needed [155].

*Antibody-based Therapy:* The use of monoclonal antibodies in cancer treatment is an appealing concept due to the selectivity and specificity with which an antibody can bind to the molecule of interest. Molecules participating in tumor progression can be targeted by antibodies through three general mechanisms: 1) Recognition of specific tumor-associated receptors, such as EGFR; 2) Binding to immune effector cells, and 3) Binding to tumor-promoting molecules such as VEGF. Though no monoclonal antibodies have been approved for the treatment of cervical cancer, researchers are accruing more convincing evidence of their value [156]. As with most cancers, cervical carcinomas possess a dynamic vascular network. Thus, much investigation has gone into developing biologic agents that target molecular pathways associated with vascularization, such as those involving vascular endothelial growth factor (VEGF). Bevaci‐ zumab is an angiogenesis inhibitor that ultimately delays vasculogenic processes, and has long been used effectively in the treatment of other malignancies such as colorectal cancer. The discovery of Bevacizumab's anti-angiogenic properties in recurrent cervical cancer during a phase II clinical trial has now warranted further investigation within the context of both singleagent and combination therapies [157]. Another antibody, Cetuximab, has a high affinity for epidermal growth factor receptor (EGFR), which is influential in cell differentiation processes. However, Cetuximab has distinguished itself as effective only against growths of squamous cell origin. Thus, other EGFR inhibitors such as gefitinib, erlotinib, and lapatinib are being investigated [158, 159]. In one research model, Cetuximab was shown to inhibit tumor cell growth following exposure to ionizing radiation, which induced EGFR pathway activation and VEGF over-expression [160]. Though others have reported Cetuximab to be more limited in activity in some populations [158], the roles of both VEGF and EGFR in cervical cancer remain under intense study. Another unique approach in helping to further define molecular targets using antibodies is to structure them against a particular domain of an HPV oncopro‐ tein. By designing mAbs against HPV16 E6 zinc-binding domains researchers are able to map key peptide sequences, and potentially interfere with cell transformation mechanisms affecting p53 tumor suppressor levels [161].

with the bovine and human papillomaviruses may differ, the concern that Brd4 may play a key role in viral replication appears to be substantiated. Regarding PV E2, research has indicated that its N-terminal transactivation domain is quite conserved among the papillo‐ maviruses [168]. Thus, many of the properties of the PV E2 protein are likely to be shared between many PVs [166]. Origin-specific viral DNA replication is overseen by E2 once the viral helicase, E1, has been loaded successfully onto the origin of replication by E2. E2 also represses the expression of E6/E7 oncoproteins at the transcriptional level, in addition to performing other regulatory tasks. Therefore, it could be quite detrimental to the intracellular establish‐ ment of the virus, its subsequent replication and cellular transformation if the interactions between E2 and its cellular partners could be targeted. For example, while Brd4 is bound to E2, E2 is unable to engage P-TEFB, a transcription elongation factor, and this affects the expression of downstream genes such as E6 and E7 from the integrated viral genome [169]. Future studies are expected to provide more conclusive data regarding P-TEFB, the roles of Brd4, and their association with HPV proteins. But as a key regulatory protein, the importance

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of E2 on HPV viability and replication makes it a prime target for intervention.

E1-inhibitor complexes.

E1 is the only enzymatic product of the viral genome, coding for an ATPase, and is thus an appealing target for molecular intervention. Indeed, if E1's binding and helicase properties could be blocked, DNA replication would be halted. Inevitably, impeding this process would also thwart the hijacking of cellular replication machinery for viral genome multiplication. Because the virus uses cellular replication factors derived from the host, current antiviral agents that block viral proteases and polymerases are ineffective in opposing HPV. DNA helicase unwinding is powered by the energy provided through ATP hydrolysis *via* the ATPase. ATP acts not only as the substrate, but also as an E1-E2 allosteric modulator [170]. As such, inhibitors sensitive to ATP concentrations, such as biphenylsulfonacetic acid, seem quite promising, in part because this agent does not directly bind ATP. Adding amides to certain positions of the biphenyl group enhances the compound's affinity for HPV6 E1, increasing its specificity [171]. However, whether biphenyl inhibitors can be applied to other HPV types remains undetermined. Furthermore, researchers have struggled to demonstrate inhibitory activity in cell-based assays [172]. In summary, future small inhibitors of E1 must directly target the enzyme's binding pocket, thereby conferring greater binding strength and specificity to

Small molecular inhibitors called indandiones are recognized as the first class of molecules to block HPV DNA replication by interrupting E1-E2 binding. The presence of indandiones induces conformational changes in E2, forming a deep binding pocket through which the small molecule modifies protein activity [173]. The success of preliminary trials attests to the great potential and need of inhibitors intended for binding pockets. Repaglinides operate similarly to indandiones in disrupting E1-E2 binding, though their effect is reversible, and they are reported to occupy a larger area of the binding pocket than do their indandione counterparts. One limitation for these classes of compounds is the poor binding frequently observed between small molecules and a large protein interface. However, these studies have demonstrated that designing small molecules to target large protein interfaces might actually be necessary in order to disclose pockets thought not to exist, or to create new ones. Another factor that must

*Small Molecule Inhibitors and Antiviral Leads:* As the field of prevention continues to advance (e.g. through the development of prophylactic vaccines), one might ask why additional resources should be directed towards the discovery of small molecule HPV inhibitors. The short answer is that due to the timeline of disease progression, it will be a few decades before these preventative measures will make a significant impact on the disease burden. In the meantime, infected women and others who do not benefit from these approaches have access to only a limited, and frequently inadequate, set of options such as lesion removal. In addition to the obvious drawbacks of surgical treatment, such as invasiveness and cytodestruction, it is well established that viral persistence is mainly responsible for disease, especially among the elderly and the immunodeficient [162]. Hence, lesions do frequently recur. In addition to these concerns, as previously stated, a low risk perception might further short circuit preven‐ tive measures, thus increasing the need to contain HPV therapeutically. Moreover, the fact that one-third of all cervical cancers are caused by types of HPV that are not included in the current vaccines [163] should maintain a sense of urgency with regards to developing more compre‐ hensive and long-lasting approaches. Though no small molecule inhibitor of HPV has yet been approved, a significant amount of antiviral agent research has focused on five major potential targets for intervention: 1) Inhibition of E1/E2 interactions, 2) E6 and E7 oncoprotein blockade, 3) Direct interference with E6AP-mediated p53 degradation, 4) Interference with interactions between HPV and other apoptotic factors (i.e. Bax and FADD), and 5) Stalling the ubiquitin proteasome system to reduce the degradation of anti-tumor proteins. The following sections will discuss these topics in greater detail.

Studies have revealed that certain host proteins are co-opted by the virus and used to carry out viral functions. For instance, the bromodomain protein, Brd4, which normally serves as a regulator of cell growth and transcription, has been implicated in the tethering of bovine papillomavirus (BPV) episomes to chromosomes in dividing cells [164, 165]. Also, it was recently published that Brd4 not only binds to the HPV regulatory protein, E2, aiding in many of its functions, but also stabilizes it [166, 167]. Although the ways in which Brd4 can interact with the bovine and human papillomaviruses may differ, the concern that Brd4 may play a key role in viral replication appears to be substantiated. Regarding PV E2, research has indicated that its N-terminal transactivation domain is quite conserved among the papillo‐ maviruses [168]. Thus, many of the properties of the PV E2 protein are likely to be shared between many PVs [166]. Origin-specific viral DNA replication is overseen by E2 once the viral helicase, E1, has been loaded successfully onto the origin of replication by E2. E2 also represses the expression of E6/E7 oncoproteins at the transcriptional level, in addition to performing other regulatory tasks. Therefore, it could be quite detrimental to the intracellular establish‐ ment of the virus, its subsequent replication and cellular transformation if the interactions between E2 and its cellular partners could be targeted. For example, while Brd4 is bound to E2, E2 is unable to engage P-TEFB, a transcription elongation factor, and this affects the expression of downstream genes such as E6 and E7 from the integrated viral genome [169]. Future studies are expected to provide more conclusive data regarding P-TEFB, the roles of Brd4, and their association with HPV proteins. But as a key regulatory protein, the importance of E2 on HPV viability and replication makes it a prime target for intervention.

discovery of Bevacizumab's anti-angiogenic properties in recurrent cervical cancer during a phase II clinical trial has now warranted further investigation within the context of both singleagent and combination therapies [157]. Another antibody, Cetuximab, has a high affinity for epidermal growth factor receptor (EGFR), which is influential in cell differentiation processes. However, Cetuximab has distinguished itself as effective only against growths of squamous cell origin. Thus, other EGFR inhibitors such as gefitinib, erlotinib, and lapatinib are being investigated [158, 159]. In one research model, Cetuximab was shown to inhibit tumor cell growth following exposure to ionizing radiation, which induced EGFR pathway activation and VEGF over-expression [160]. Though others have reported Cetuximab to be more limited in activity in some populations [158], the roles of both VEGF and EGFR in cervical cancer remain under intense study. Another unique approach in helping to further define molecular targets using antibodies is to structure them against a particular domain of an HPV oncopro‐ tein. By designing mAbs against HPV16 E6 zinc-binding domains researchers are able to map key peptide sequences, and potentially interfere with cell transformation mechanisms

304 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

*Small Molecule Inhibitors and Antiviral Leads:* As the field of prevention continues to advance (e.g. through the development of prophylactic vaccines), one might ask why additional resources should be directed towards the discovery of small molecule HPV inhibitors. The short answer is that due to the timeline of disease progression, it will be a few decades before these preventative measures will make a significant impact on the disease burden. In the meantime, infected women and others who do not benefit from these approaches have access to only a limited, and frequently inadequate, set of options such as lesion removal. In addition to the obvious drawbacks of surgical treatment, such as invasiveness and cytodestruction, it is well established that viral persistence is mainly responsible for disease, especially among the elderly and the immunodeficient [162]. Hence, lesions do frequently recur. In addition to these concerns, as previously stated, a low risk perception might further short circuit preven‐ tive measures, thus increasing the need to contain HPV therapeutically. Moreover, the fact that one-third of all cervical cancers are caused by types of HPV that are not included in the current vaccines [163] should maintain a sense of urgency with regards to developing more compre‐ hensive and long-lasting approaches. Though no small molecule inhibitor of HPV has yet been approved, a significant amount of antiviral agent research has focused on five major potential targets for intervention: 1) Inhibition of E1/E2 interactions, 2) E6 and E7 oncoprotein blockade, 3) Direct interference with E6AP-mediated p53 degradation, 4) Interference with interactions between HPV and other apoptotic factors (i.e. Bax and FADD), and 5) Stalling the ubiquitin proteasome system to reduce the degradation of anti-tumor proteins. The following sections

Studies have revealed that certain host proteins are co-opted by the virus and used to carry out viral functions. For instance, the bromodomain protein, Brd4, which normally serves as a regulator of cell growth and transcription, has been implicated in the tethering of bovine papillomavirus (BPV) episomes to chromosomes in dividing cells [164, 165]. Also, it was recently published that Brd4 not only binds to the HPV regulatory protein, E2, aiding in many of its functions, but also stabilizes it [166, 167]. Although the ways in which Brd4 can interact

affecting p53 tumor suppressor levels [161].

will discuss these topics in greater detail.

E1 is the only enzymatic product of the viral genome, coding for an ATPase, and is thus an appealing target for molecular intervention. Indeed, if E1's binding and helicase properties could be blocked, DNA replication would be halted. Inevitably, impeding this process would also thwart the hijacking of cellular replication machinery for viral genome multiplication. Because the virus uses cellular replication factors derived from the host, current antiviral agents that block viral proteases and polymerases are ineffective in opposing HPV. DNA helicase unwinding is powered by the energy provided through ATP hydrolysis *via* the ATPase. ATP acts not only as the substrate, but also as an E1-E2 allosteric modulator [170]. As such, inhibitors sensitive to ATP concentrations, such as biphenylsulfonacetic acid, seem quite promising, in part because this agent does not directly bind ATP. Adding amides to certain positions of the biphenyl group enhances the compound's affinity for HPV6 E1, increasing its specificity [171]. However, whether biphenyl inhibitors can be applied to other HPV types remains undetermined. Furthermore, researchers have struggled to demonstrate inhibitory activity in cell-based assays [172]. In summary, future small inhibitors of E1 must directly target the enzyme's binding pocket, thereby conferring greater binding strength and specificity to E1-inhibitor complexes.

Small molecular inhibitors called indandiones are recognized as the first class of molecules to block HPV DNA replication by interrupting E1-E2 binding. The presence of indandiones induces conformational changes in E2, forming a deep binding pocket through which the small molecule modifies protein activity [173]. The success of preliminary trials attests to the great potential and need of inhibitors intended for binding pockets. Repaglinides operate similarly to indandiones in disrupting E1-E2 binding, though their effect is reversible, and they are reported to occupy a larger area of the binding pocket than do their indandione counterparts. One limitation for these classes of compounds is the poor binding frequently observed between small molecules and a large protein interface. However, these studies have demonstrated that designing small molecules to target large protein interfaces might actually be necessary in order to disclose pockets thought not to exist, or to create new ones. Another factor that must be considered is the fact that viral integration into the host genome frequently leads to loss of E1/E2 gene expression, meaning that established cancers are likely to have lost the molecules targeted by inhibitors of E1 and/or E2, thereby limiting their usefulness [174, 175].

most attention. In one such study, small molecules were screened and selected based on their MDM2inhibitoryproperties,andaclasscalledtheNutlinswasdiscovered.Nutlinscompetitive‐ ly bind MDM2 at the same site typically occupied by p53, and structurally interpose them‐ selves between p53 and MDM2 [185]. In contrast, another molecule labeled RITA actually binds to p53 and stabilizes it against degradation by inhibiting p53 from interacting with most of its binding partners, including MDM2 [186]. A more recent addition to the MDM2 inhibitor group is TRIAD1, a RING-finger bearing molecule, that functions similarly to RITA in that it binds p53

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One well-established inhibitor of the UPS is Bortezomib. Bortezomib targets and reversibly blocks26Sproteasomeactivity,andhasalreadybeenFDA-approvedforthetreatmentofmultiple myeloma and lymphoma [188]. Though its use has been proposed for the treatment of many diseases, from non-small cell lung cancer to pancreatic cancer, an equivalent and thorough exploration in the context of cervical carcinoma is still needed [189]. This suggestion is solidly founded on the observed sensitization of cervical cancer cells to apoptosis by another protease inhibitor (PI), MG132 [190]. A final set of PIs are those that inhibit the HIV protease. The antioncogenicproperties of HIVPIs were firstnoted withrespectto the 20Sproteasome, andfurther investigation explicitly demonstrated Lopinavir active against E6-induced p53 degradation. Though Lopinavir also stabilizes p53, it exhibits low potency and virus is not fully cleared. The value of HIV PIs in cervical cancer treatment could be potentiated by its current availability as

While p53 and the proteins to which it is connected are clearly targets worth exploring, other pro-apoptotic targets could prove just as important in halting the progression of HPV-mediat‐ eddisease.HPV16E6bindstoseveraladditionalsignalingmoleculesintheintrinsicandextrinsic apoptotic pathways, including Bax, FADD, and procaspase-8, thus blocking their ability to interact with their normal partners and leading to their premature disposal by the protea‐ some. Not only does HPV16 E6 indirectly affect Bax *via* the degradation of p53, but Bax mRNA levels are decreased and the protein itself is destabilized in the presence of E6. Therefore, apoptotic cascades involvingBax andp53 represent a compelling site at whichantiviraltherapy could be targeted [194, 195]. It has also been reported that HPV16 E6 binds to both FADD and caspase 8 *via* DED residues, and a peptide corresponding to the binding site of FADD blocked both of these interactions. Expression of this peptide in HPV+ cells was able to re-sensitize those cellstoapoptosistriggeredthroughtheextrinsicpathway[196,197].Asearchforsmallmolecules capable of interfering with these interactions was conducted and several candidates were identified, primarily among the flavones and flavonols. Of these compounds, myricetin generatedthelowestIC50 inassaysdesignedtodetectinhibitionofE6-procaspase8binding[198].

In summary, the scientific community has witnessed tremendous progress in the recent years towards the goal of eradicating HPV-mediated cervical carcinoma. Of these endeavors, routine Pap testing and the prophylactic vaccines, Gardasil and Cervarix, are particularly noteworthy

(at the C-terminus), and also intercepts ubiquitination triggered by MDM2 [187].

an antiviral agent, which might expedite the clinical trial process [191-193].

More research is needed to optimize and test these small molecule leads.

**5. Final remarks**

In contrast, E6 and E7 are frequently over-expressed in established cancers, making these two proteins quite attractive as targets. E6 and E7 are the zinc finger-containing proteins primarily responsible for the malignant alterations and de-differentiation of keratinocytes observed during cell transformation. These changes occur following integration of the HPV genome into host DNA [163, 176]. During this process, the regulators of viral replication, E1 and E2, are frequently disrupted, allowing over-expression of E6 and E7. HR-HPV types induce cell immortalization and transformation primarily through the over-expression of E6 and/or E7, which are best known for their ability to accelerate the degradation of the p53 and retinoblas‐ toma proteins (pRB), respectively. The E6-mediated loss of p53 function leads to an insensi‐ tivity to apoptotic signals as well as to a loss of cell cycle regulation at the G1/S checkpoint in response to DNA damage. E7 contributes to the hyperplasia crisis by accelerating the degra‐ dation of pRB and thereby stimulating cells in Interphase to re-enter the cell cycle at S phase [177-179]. Together, over-expression of the E6 and E7 oncoproteins, decrease apoptosis and increase cell division, setting the stage for cancer [180]. Antiviral agents that can partially, if not fully, inhibit E6 and/or E7 functions clearly have the potential to negatively impact the carcinogenic process. One group, for example, proposed such a strategy in their study of the HPV16 E7-antagonizing peptide, Pep-7 [181]. Pep-7 was originally introduced as a short peptide component of the vacuole/lysosomal pathway [182]. However, Pep-7 was later shown not only to reduce the viability of HPV-positive cells *in vitro*, but it also decreased expression of E7 in SiHa cells in a xenograft model. It is conjectured that the selective mechanism Pep-7 uses to suppress cell proliferation may hinge on its ability to obstruct E7-pRB associations, even releasing pRB from E7 [181].

In contrast to E7, which appears to act primarily by increasing the ability of expressing cells to replicate, E6 acts by reducing the ability of expressing cells to undergo apoptosis. Apoptosis is a natural, cell-mediated death response to irreparable DNA damage. One target of E6 is the p53 tumor suppressor, which is degraded following association of E6 with the ubiquitin protein ligase, E6AP. The E6/E6AP complex binds to p53 and initiates its ubiquitination and conse‐ quent proteolytic destruction [183]. This means that the downstream targets of p53, which mediate cell cycle arrest and apoptosis, are not activated. Therefore, interference with the E6/ E6AP-mediated proteasomal degradation of p53 has been seen as another possible strategy for treatment.Theubiquitinationproteasome system (UPS) begins with theubiquitin activatingE1 molecules interactingwithE2conjugatingenzymes,followedbycatalyzationofthepolyubiqui‐ tination cascade onto target proteins by E3 enzymes [184]. A subset of E3s, called RING-finger E3s, are a group of ubiquitin ligases that have domains to which ubiquitination substrates bind, anditis thoughtthat by inhibiting this interaction,p53might bepreserved.Oneprominentp53 related RING-finger ubiquitin ligase is MDM2. MDM2 is normally expressed in a negative feedback manner to regulate p53 levels. Three dominant trains of thought have guided approaches seeking ways in which the negative effects of MDM2 might be neutralized: 1) Blocking activation domains on p53, 2) Increasing nuclear export of p53 so as not to activate MDM2 transcription, and 3) Inhibiting MDM2. Of these, the third approach has received the

most attention. In one such study, small molecules were screened and selected based on their MDM2inhibitoryproperties,andaclasscalledtheNutlinswasdiscovered.Nutlinscompetitive‐ ly bind MDM2 at the same site typically occupied by p53, and structurally interpose them‐ selves between p53 and MDM2 [185]. In contrast, another molecule labeled RITA actually binds to p53 and stabilizes it against degradation by inhibiting p53 from interacting with most of its binding partners, including MDM2 [186]. A more recent addition to the MDM2 inhibitor group is TRIAD1, a RING-finger bearing molecule, that functions similarly to RITA in that it binds p53 (at the C-terminus), and also intercepts ubiquitination triggered by MDM2 [187].

One well-established inhibitor of the UPS is Bortezomib. Bortezomib targets and reversibly blocks26Sproteasomeactivity,andhasalreadybeenFDA-approvedforthetreatmentofmultiple myeloma and lymphoma [188]. Though its use has been proposed for the treatment of many diseases, from non-small cell lung cancer to pancreatic cancer, an equivalent and thorough exploration in the context of cervical carcinoma is still needed [189]. This suggestion is solidly founded on the observed sensitization of cervical cancer cells to apoptosis by another protease inhibitor (PI), MG132 [190]. A final set of PIs are those that inhibit the HIV protease. The antioncogenicproperties of HIVPIs were firstnoted withrespectto the 20Sproteasome, andfurther investigation explicitly demonstrated Lopinavir active against E6-induced p53 degradation. Though Lopinavir also stabilizes p53, it exhibits low potency and virus is not fully cleared. The value of HIV PIs in cervical cancer treatment could be potentiated by its current availability as an antiviral agent, which might expedite the clinical trial process [191-193].

While p53 and the proteins to which it is connected are clearly targets worth exploring, other pro-apoptotic targets could prove just as important in halting the progression of HPV-mediat‐ eddisease.HPV16E6bindstoseveraladditionalsignalingmoleculesintheintrinsicandextrinsic apoptotic pathways, including Bax, FADD, and procaspase-8, thus blocking their ability to interact with their normal partners and leading to their premature disposal by the protea‐ some. Not only does HPV16 E6 indirectly affect Bax *via* the degradation of p53, but Bax mRNA levels are decreased and the protein itself is destabilized in the presence of E6. Therefore, apoptotic cascades involvingBax andp53 represent a compelling site at whichantiviraltherapy could be targeted [194, 195]. It has also been reported that HPV16 E6 binds to both FADD and caspase 8 *via* DED residues, and a peptide corresponding to the binding site of FADD blocked both of these interactions. Expression of this peptide in HPV+ cells was able to re-sensitize those cellstoapoptosistriggeredthroughtheextrinsicpathway[196,197].Asearchforsmallmolecules capable of interfering with these interactions was conducted and several candidates were identified, primarily among the flavones and flavonols. Of these compounds, myricetin generatedthelowestIC50 inassaysdesignedtodetectinhibitionofE6-procaspase8binding[198]. More research is needed to optimize and test these small molecule leads.
