The Inhibitory Effect of KerraTM, KSTM, and MinozaTM on Human Papillomavirus Infection and Cervical Cancer.

Background and Objectives: Cervical cancer is one of the most common types of frequently found cancers in Thailand. One of the causative agents is the infection of the high-risk human papillomavirus (HPV) type 16 and 18. Traditional medicines are rich sources of bioactive compounds which are a valuable source for the development of novel cancer therapies. In this study, the therapeutic effects of 3 traditional medicines, KerraTM, KSTM, and MinozaTM, were studied on HeLa and CaSki cells. Materials and Methods: The effects of KerraTM, KSTM, and MinozaTM on cancer cells were evaluated through cytotoxicity and cell death assays. The infection assay using HPV-16 pseudovirus was also carried out. Results: All traditional medicines efficiently suppressed cell growths of HeLa and CaSki, with KerraTM being the most potent anticancer agent followed by KSTM and MinozaTM. KerraTM at 158 µg/mL and 261 µg/mL significantly increases the percentage inhibition of the HPV-16 pseudovirus infection in a pre-attachment step in a dose-dependent manner, while KSTM at 261 µg/mL efficiently inhibited viral infection in both pre-attachment and adsorption steps. However, KerraTM, KSTM, and MinozaTM at subtoxic concentrations could not reduce the viral E6 mRNA expressions of HPV-16 and HPV-18. Cell death assay by acridine orange/ethidium bromide showed that KerraTM increased population of dead cells in dose-dependent manner in both CaSki and HeLa. The percentage of secondary necrosis in KerraTM-treated CaSki was higher than that of HeLa cells, while the percentage of late apoptotic cells in HeLa was higher than that of CaSki, indicating that HeLa was more susceptible to KerraTM than CaSki. For KSTM and MinozaTM, these extracts at 250 µg/mL promoted autophagy over cell death. At 500 µg/mL, the percentage of dead cells in KerraTM was higher than that of KSTM and MinozaTM. Conclusions: KerraTM is a potent traditional medicine for promoting cancer cell death. KerraTM is possibly useful in the prevention and treatment of cervical cancer. Further investigation will be carried out to gain a better understanding of the biochemical mechanism and the pharmacological activity underlying this effect.

Green Tea Polyphenols Cause Apoptosis and Autophagy in HPV-16 Subgene-Immortalized Human Cervical Epithelial Cells via the Activation of the Nrf2 Pathway.

Infection with human papillomavirus (HPV) is relatively common and certain high-risk HPV strains can induce epithelial dysplasia, increasing the risk of cervical cancer. Green tea polyphenol (GTP) preparations exhibit diverse anti-inflammatory, antioxidative, and antitumor properties In Vitro and In Vivo. Topical GTP application has been recommended as a treatment for genital warts, but the effect of GTP treatment on HPV infection and HPV-associated cancer remains to be established. The present study aimed to explore the mechanism by which GTP affected HPV type 16 (HPV-16)-positive immortalized human cervical epithelial cells. Survival, apoptosis, and autophagocytosis of these cells following GTP treatment was assessed using CCK-8 assay, flow cytometry, and monodansylcadaverine (MDC) staining. These cells were further transfected with an shRNA specific for Nrf2 to generate stable Nrf2-knockdown cells. The levels of Caspase-3, Bcl-2, Bax, P53, Rb, HPV-16 E6, HPV-16 E7, P62, Beclin1 and LC3B were determined via Western blotting. These analyses revealed that GTP treatment induced autophagy and apoptosis in HPV-16-positive cells, while Nrf2 gene knockdown reversed GTP-induced autophagic and apoptotic effects. Together, these results suggested that GTP could alleviate HPV infection and HPV-associated precancerous lesions In Vitro by regulating the Nrf2 pathway, highlighting the therapeutic potential of GTP in treating HPV infection.

Broad Neutralization Responses Against Oncogenic Human Papillomaviruses Induced by a Minor Capsid L2 Polytope Genetically Incorporated Into Bacterial Ferritin Nanoparticles.

Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.

Development of HPV16,18,31,45 E5 and E7 peptides-based vaccines predicted by immunoinformatics tools.

OBJECTIVES: Viral oncoproteins are ideal targets in therapeutic vaccines for functional inhibition of human papillomaviruses (HPVs). Herein, we designed the peptide constructs derived from E5 and E7 oncoproteins of high-risk HPV types 16, 18, 31 and 45 using the bioinformatics tools and investigated their potency in mice. RESULTS: The framework of the combined in silico/in vivo analysis included (1) to determine physicochemical properties of the designed constructs, (2) to identify potential IFN-γ-inducing epitopes, (3) to assess allergenicity, (4) to recognize linear and discontinuous B cell epitopes using modeling and validation of 3D structure of the designed constructs, and (5) to evaluate immune responses and tumor growth in vivo. Our in silico data determined high potency of the HPV16,18,31,45 E5 and HPV16,18,31,45 E7 peptides for trigger B- and T-cell responses, and IFN-γ secretion. In vivo study indicated that the mixture of E5 and E7 immunodominant peptides from four types of high-risk HPV could induce Th1 immune response, and protect completely mice against TC-1 tumor cells. CONCLUSION: Generally, the combined in silico/in vivo approaches showed the ability of the designed E5 and E7 peptide constructs from four major high-risk HPV types for development of therapeutic vaccines.

The Induction of the Synthesis of Interferon, CD4 and CD8 T Lymphocytes in Blood and Spleen of Mice after Oral Vaccination with the "Early" Protein HPV16 E2.

High contents of interferon and CD4 and CD8 T lymphocytes were found in blood and spleen cells of mice after their oral vaccination with the "early" protein of HPV16 E2 by using ELISPOT, Western blot hybridization, and enzyme immunoassay technique. It was concluded that the "early" protein HPV16 E2 is involved in the immune system activation during control of pathogenesis and neoplasia.

A Novel Human Papillomavirus 16 L1 Pentamer-Loaded Hybrid Particles Vaccine System: Influence of Size on Immune Responses.

Cervical cancer remains the second-most prevalent female malignancy around the world, leading to a great majority of cancer-related mortality that occurs mainly in developing countries. Developing an effective and low-cost vaccine against human papillomavirus (HPV) infection, especially in medically underfunded areas, is urgent. Compared with vaccines based on HPV L1 viruslike particles (VLPs) in the market, recombinant HPV L1 pentamer expressed in Escherichia coli represents a promising and potentially cost-effective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have shown great potential compared to conventional aluminum salts adjuvant and is urgently needed for HPV L1 pentamer vaccines. It is well-reported that particle sizes are crucial in regulating immune responses. Nevertheless, reports on the relationship between the particulate size and the resultant immune response have been in conflict, and there is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Here, we fabricated HPV 16 L1 pentamer-loaded poly(d,l-lactide- co-glycolide) (PLGA)/lecithin hybrid particles with uniform sizes (0.3, 1, and 3 µm) and investigated the particle size effects on antigen release, activation of lymphocytes, dendritic cells (DCs) activation and maturation, follicular helper CD4+ T (TFH) cells differentiation, and release of pro-inflammatory cytokines and chemokines. Compared with the other particle sizes, 1 µm particles induced more powerful antibody protection and yielded more persistent antibody responses, as well as more heightened anamnestic responses upon repeat vaccination. The superior immune responses might be attributed to sustainable antigen release and robust antigen uptake and transport and then further promoted a series of cascade reactions, including enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in draining lymph nodes (DLNs). Here, a powerful and economical platform for HPV vaccine and a comprehensive understanding of particle size effect on immune responses for HPV L1 pentamer-based candidate vaccines are provided.

Human papilloma virus-specific T cells can be generated from naïve T cells for use as an immunotherapeutic strategy for immunocompromised patients.

Human papilloma virus (HPV) is a known cause of cervical cancer, squamous cell carcinoma and laryngeal cancer. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T-cell immunity is essential for disease control. Because Epstein-Barr Virus (EBV)-specific T cells have been highly successful in treating or preventing EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third-party setting to treat patients with high-risk/relapsed HPV-associated cancers. Most protocols for generating virus-specific T cells require prior exposure of the donor to the targeted virus and, because the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells has proven challenging. We, therefore, made systematic changes to our current Good Manufacturing Practice (GMP)-compliant protocols to improve antigen presentation, priming and expansion for the manufacture of high-efficacy HPV-specific T cells. Like others, we found that current methodologies fail to expand HPV-specific T cells from most healthy donors. By optimizing dendritic cell maturation and function with lipopolysaccharide (LPS) and interferon (IFN)γ, adding interleukin (IL)-21 during priming and depleting memory T cells, we achieved reliable expansion of T cells specific for oncoproteins E6 and E7 to clinically relevant amounts (mean, 578-fold expansion; n = 10), which were polyfunctional based on cytokine multiplex analysis. In the third-party setting, such HPV-specific T-cell products might serve as a potent salvage therapy for patients with HPV-associated diseases.

HPV16 E6 Promotes Breast Cancer Proliferation via Upregulation of COX-2 Expression.

Background. Breast cancer remains the leading cause of cancer-related mortality worldwide. It has been indicated that human papillomaviruses 16 (HPV16) might participate in the pathogenesis and development of breast cancer. However, the detected rate of HPV16 varies with region. We will investigate HPV16 E6 expression in North China and explore the effects and mechanism of HPV16 E6 on breast cancer proliferation in this study. Methods. The expressions of HPV16 E6 and COX-2 in paraffin-embedded tissues of the invasive ductal breast cancer were detected by qPCR and IHC. The effects of HPV16 E6 on breast cancer proliferation were determined by function studies. The mechanism of HPV16 E6 in promoting breast cancer proliferation was explored by Western blot and Dual-Luciferase Reporter Assay. Results. HPV16 E6 was positive in 28% invasive ductal breast carcinoma in North China; HPV16 E6 promoted breast cancer proliferation. Inhibition of COX-2 by siCOX-2 or Celecoxib attenuated the proliferation of breast cancer cells with HPV16 E6 expression; and the upregulation of COX-2 could be suppressed by the inhibition of NF-κB activity. Conclusion. HPV16 E6 promotes breast cancer proliferation by activation of NF-κB signaling pathway and increase of COX-2 expression. COX-2 will be a potential target for HPV16 E6-associated breast cancer.

TLR2 ligand-synthetic long peptide conjugates effectively stimulate tumor-draining lymph node T cells of cervical cancer patients.

The potency of human papillomavirus type 16 (HPV16)-encoded synthetic long peptides (SLP), conjugated to an optimized Toll-like receptor 2 ligand (TLR2-L), was assessed in ex vivo activation of HPV16+ cancer patient-derived T cells. Two highly immunogenic SLP sequences derived from the oncogenic E6 protein of HPV16 were selected and conjugated to a Pam3CSK4-based TLR2-L under GMP conditions. Both conjugates were able to mature human DCs in vitro and to activate human skin-derived antigen-presenting cells (APCs) upon intradermal injection in an ex vivo skin model, associated with induction of a favorable chemokine profile to attract and activate T cells. The conjugated SLPs were efficiently processed by APCs, since HPV16-specific CD4+ and CD8+ T-cell clones isolated from HPV16+ cervical tumors proliferated in response to both conjugates. The TLR2-L SLP conjugates significantly enhanced ex vivo T helper type 1 T-cell activation in cell suspensions obtained from tumor-draining lymph nodes (LN) resected during hysterectomy of HPV16+ cervical cancer patients. These results show that TLR2-L SLP conjugates can activate circulating or LN-derived tumor-specific T cells, a promising outcome for studying these two conjugates in a phase I/II clinical safety and immunogenicity trial.

Peptiderive server: derive peptide inhibitors from protein-protein interactions.

The Rosetta Peptiderive protocol identifies, in a given structure of a protein-protein interaction, the linear polypeptide segment suggested to contribute most to binding energy. Interactions that feature a 'hot segment', a linear peptide with significant binding energy compared to that of the complex, may be amenable for inhibition and the peptide sequence and structure derived from the interaction provide a starting point for rational drug design. Here we present a web server for Peptiderive, which is incorporated within the ROSIE web interface for Rosetta protocols. A new feature of the protocol also evaluates whether derived peptides are good candidates for cyclization. Fast computation times and clear visualization allow users to quickly assess the interaction of interest. The Peptiderive server is available for free use at http://rosie.rosettacommons.org/peptiderive.

Integrating transcriptional and protein interaction networks to prioritize condition-specific master regulators.

BACKGROUND: Genome-wide libraries of yeast deletion strains have been used to screen for genes that drive phenotypes such as stress response. A surprising observation emerging from these studies is that the genes with the largest changes in mRNA expression during a state transition are not those that drive that transition. Here, we show that integrating gene expression data with context-independent protein interaction networks can help prioritize master regulators that drive biological phenotypes. RESULTS: Genes essential for survival had previously been shown to exhibit high centrality in protein interaction networks. However, the set of genes that drive growth in any specific condition is highly context-dependent. We inferred regulatory networks from gene expression data and transcription factor binding motifs in Saccharomyces cerevisiae, and found that high-degree nodes in regulatory networks are enriched for transcription factors that drive the corresponding phenotypes. We then found that using a metric combining protein interaction and transcriptional networks improved the enrichment for drivers in many of the contexts we examined. We applied this principle to a dataset of gene expression in normal human fibroblasts expressing a panel of viral oncogenes. We integrated regulatory interactions inferred from this data with a database of yeast two-hybrid protein interactions and ranked 571 human transcription factors by their combined network score. The ranked list was significantly enriched in known cancer genes that could not be found by standard differential expression or enrichment analyses. CONCLUSIONS: There has been increasing recognition that network-based approaches can provide insight into critical cellular elements that help define phenotypic state. Our analysis suggests that no one network, based on a single data type, captures the full spectrum of interactions. Greater insight can instead be gained by exploring multiple independent networks and by choosing an appropriate metric on each network. Moreover we can improve our ability to rank phenotypic drivers by combining the information from individual networks. We propose that such integrative network analysis could be used to combine clinical gene expression data with interaction databases to prioritize patient- and disease-specific therapeutic targets.

E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response.

Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.

Upregulation of p18Ink4c expression by oncogenic HPV E6 via p53-miR-34a pathway.

Binding of p53 to miR-34a promoter activates the expression of tumor-suppressive miR-34a. Oncogenic human papillomavirus (HPV) infection downregulates miR-34a expression through viral E6 degradation of p53. In our report, we found that miR-34a specifically targets p18Ink4c, a CDK4 and CDK6 inhibitor induced by E2F transactivation. HPV18(+) HeLa cells with ectopic miR-34a expression or by E6 siRNA knockdown-induced expression of endogenous miR-34a exhibited a substantial reduction of p18Ink4c in a dose-dependent manner, but had no effect on p16Ink4a, another member of CDK4/6 inhibitor family. In contrast, de novo infection by oncogenic HPVs of human keratinocyte-derived raft tissues increased p18Ink4c expression. Suppression of endogenous miR-34a in cell lines with a miR-34a inhibitor also increased p18Ink4c. We found that miR-34a suppresses the expression of p18Ink4c by binding to a specific seed match in the 5' UTR of p18Ink4c. Further investigation found remarkable increase of p18Ink4c in cervical precancer lesions and cervical cancer. Immunohistochemical staining of cervical tissue arrays showed increased expression of p18Ink4c in 68% of cervical cancer, 8.3% of chronic cervical inflammation and 4.8% of normal cervix. Although p18Ink4c inhibits cell proliferation in general and regulates E2F1 expression in HCT116 cells, it appears not to function as a tumor suppressor in cervical cancer cells lacking an intact G1 checkpoint because of viral E7 degradation of pRB. In summary, our study demonstrates an intimate connection among oncogenic HPV E6, p53, miR-34a and p18Ink4c and identifies p18Ink4c as a possible biomarker for cervical cancer.

Up-regulation of interleukin-17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer.

BACKGROUND: Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)-17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line. METHODS: IL-17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL-17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6-transfected H1299 human NSCLC cells. RESULTS: Immunohistochemical data showed that 48.1% of lung tumors had IL-17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL-17 with IL-6 (P < .001) and IL-17 with Mcl-1 (P < .001) expression were also observed. Cell growth rate was increased, and IL-17/Mcl-1 expression levels were elevated in HPV 16 E6-transfected H1299 cells. The transfected E6 oncoproteins can significantly up-regulate expression levels of IL-17 and antiapoptotic protein Mcl-1. CONCLUSIONS: The study suggests that HPV infection-induced IL-17 levels can stimulate Mcl-1 expression through the PI3K pathway and promote lung tumor cell progression through a p53- and IL-6-independent pathway.

Influenza virus activates inflammasomes via its intracellular M2 ion channel.

Influenza virus, a negative-stranded RNA virus that causes severe illness in humans and animals, stimulates the inflammasome through the Nod-like receptor NLRP3. However, the mechanism by which influenza virus activates the NLRP3 inflammasome is unknown. Here we show that the influenza virus M2 protein, a proton-selective ion channel important in viral pathogenesis, stimulates the NLRP3 inflammasome pathway. M2 channel activity was required for the activation of inflammasomes by influenza and was sufficient to activate inflammasomes in primed macrophages and dendritic cells. M2-induced activation of inflammasomes required its localization to the Golgi apparatus and was dependent on the pH gradient. Our results show a mechanism by which influenza virus infection activates inflammasomes and identify the sensing of disturbances in intracellular ionic concentrations as a previously unknown pathogen-recognition pathway.

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor.

This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.

Addition of a single E2 binding site to the human papillomavirus (HPV) type 16 long control region enhances killing of HPV positive cells via HPV E2 protein-regulated herpes simplex virus type 1 thymidine kinase-mediated suicide gene therapy.

Human papillomavirus type 16 (HPV16) is associated with the development of anogenital cancers and their precursor lesions, intraepithelial neoplasia. Treatment strategies against HPV-induced intraepithelial neoplasia are not HPV specific and mostly consist of physical removal or ablation of lesions. We had previously designed an HPV-specific approach to kill HPV-infected cells by the herpes simplex virus type 1 thymidine kinase (TK) gene driven by HPV E2 binding to E2-binding sites (E2BS) in the native HPV16 long control region. E2-induced TK expression renders the cells sensitive to the prodrug ganciclovir. To optimize this therapeutic approach, we modified the native long control region by adding variable numbers of E2BS adjacent to E2BS4, resulting in greatly increased cell death in HPV-positive cell lines with variable levels of E2 protein expression and no reduction in HPV specificity. Our results showed maximum increase in TK expression and cell killing when one additional E2BS was added adjacent to E2BS. As HPV-infected patients also exhibit variable E2 expression across lesions and within a lesion, this approach may potentiate the clinical utility of the herpes simplex virus type 1 TK/ganciclovir therapeutic approach.

HMGB2 stabilizes p53 by interfering with E6/E6AP-mediated p53 degradation in human papillomavirus-positive HeLa cells.

We investigated the effect of HMGB2 on the stability of p53 protein in HeLa cells. Overexpression of HMGB2 led to accumulation of the p53 protein, whereas HMGB2 knockdown with siRNA resulted in a substantial decrease in the p53 protein level. The HMGB2-dependent increase of p53 stability was specific for HPV-positive HeLa cells as HCT116 and MCF7 cell lines did not demonstrate this response. Co-expression of HMGB2 and HPV E6 prevented HPV E6 protein-mediated ubiquitination and degradation of p53. FACS analysis exhibited that HeLa cells transfected with HMGB2 displayed decreased cell proliferation, with a concomitant increase of the p53 protein and arrest of the cell cycle, predominantly in G1 phase. Our findings collectively suggest that HMGB2 could stabilize p53 by interfering with E6/E6AP-mediated p53 degradation in HPV-positive HeLa cells.

Kinase requirements in human cells: II. Genetic interaction screens identify kinase requirements following HPV16 E7 expression in cancer cells.

Human papillomavirus (HPV) oncoproteins subvert cellular signaling pathways, including kinase pathways, during the carcinogenic process. To identify kinases targeted by the HPV16 E7 oncoprotein, shRNA kinase screens were performed in RKO colorectal carcinoma cell lines that differ only in their expression of HPV16 E7. Our screens identified kinases that were essential for the survival of RKO cells, but not essential for RKO cells expressing HPV16 E7. These kinases include CDK6, ERBB3, FYN, AAK1, and TSSK2. We show that, as predicted, CDK6 knockdown inhibits pRb phosphorylation and induces S-phase depletion, thereby inhibiting cell viability. Knockdown of ERBB3, FYN, AAK1, and TSSK2 induces a similar loss of cell viability through an unknown mechanism. Expression of the HPV16 E7 oncoprotein, known to bind and degrade pRb, relieves the requirement of these kinases. These studies demonstate that expression of a single oncoprotein can dramatically alter kinase sensitivity in human cells. The shRNA screens used here perform analogously to genetic interaction screens commonly used in genetically tractable organisms such as yeast, and thus represent an exciting method for unbiased identification of cellular signaling pathways targeted by cancer mutations.

Reactive oxygen species play a central role in the activity of cationic liposome based cancer vaccine.

Recently, we developed a simple and potent therapeutic liposome cancer vaccine consisting of a peptide antigen and a cationic lipid. The molecular mechanism of the adjuvanticity of cationic liposome was studied and described in the current report. First, cationic DOTAP liposome, but not the neutral liposome DOPC, was shown to generate reactive oxygen species (ROS) in mouse bone marrow-derived dendritic cells (BMDC). ROS generation by DOTAP was required for ERK and p38 activation and downstream chemokine/cytokine induction. Furthermore, ROS were shown to be involved in the expression of the co-stimulatory molecules CD86/CD80 induced by DOTAP. However, as the DOTAP concentration increased from 50 to 800 microM, the apoptotic marker Annexin V and ROS double positive cells increased, suggesting that high dose of DOTAP-generated ROS causes cell apoptosis. In vivo, optimal amount of ROS in the draining lymph nodes (DLN) and anti-tumor (HPV positive TC-1 tumor) activity induced by E7 peptide (antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16) formulated in 100 nmol DOTAP were attenuated by incorporating DOPC in the formulation, suggesting that ROS are essential for the vaccine induced anti-tumor activity. Moreover, 600 nmol DOTAP/E7 generated huge amount of ROS in the DLN and showed no activity of tumor regression. Interestingly, 600 nmol DOTAP/E7-induced ROS were tuned down to the same level induced by 100 nmol DOTAP/E7 by adding DOPC in the formulation and this formulation showed tumor regression activity. In conclusion, DOTAP is an active DC stimulator resulting in the activation of ERK and p38 and induction of chemokines, cytokines and co-stimulatory molecules mediated by appropriate amount of ROS. Our data elucidated an important mechanism of adjuvant activity of cationic liposome and could facilitate rational design of synthetic lipid based adjuvants and vaccine formulation.