Search

While both pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine and TA-HPV recombinant vaccinia viral vector-based vaccines have elicited HPV-specific CD8+ T cell responses in HPV16/E7-expressing tumor models, and been used as prime-boost regimen to enhance HPV-specific immune responses in humans (NCT00788164), the optimal route of administration for TA-HPV remains unclear. In a preclinical model, we examined the immunogenicity of priming with intramuscular pNGVL4a-Sig/E7(detox)/HSP70 followed by TA-HPV boost through different administration routes. We observed that priming twice with a pNGVL4a-Sig/E7(detox)/HSP70 followed by a single TA-HPV immunization boost through skin scarification generated the strongest antigen-specific CD8+ T cell response in C57BL/6 mice. These data translate to tumor control and prolonged survival of treated mice. Our results provide rationale for future clinical testing of intramuscular pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine prime, TA-HPV vaccine skin scarification boost immunization regimen for the control of HPV-associated diseases.

SEEK and you may find cancer earlier Many cancers can be cured by surgery and/or systemic therapies when detected before they have metastasized. This clinical reality, coupled with the growing appreciation that cancer's rapid genetic evolution limits its response to drugs, have fueled interest in methodologies for earlier detection of the disease. Cohen et al. developed a noninvasive blood test, called CancerSEEK that can detect eight common human cancer types (see the Perspective by Kalinich and Haber). The test assesses eight circulating protein biomarkers and tumor-specific mutations in circulating DNA. In a study of 1000 patients previously diagnosed with cancer and 850 healthy control individuals, CancerSEEK detected cancer with a sensitivity of 69 to 98% (depending on cancer type) and 99% specificity. Science , this issue p. 926 ; see also p. 866

Background: This study prospectively examines weight gain in breast cancer survivors compared with cancer-free women from a familial risk cohort. Methods: Absolute and percent weight change over 4 years was compared among 303 breast cancer survivors and 307 cancer-free women matched on age and menopausal status, from the same familial risk cohort. Linear and logistic regression was used to estimate the association between survivor status and weight gain. Results: Overall, breast cancer survivors gained significantly more weight [β = 3.06 pounds; 95% confidence intervals (CI), 0.94–5.17] than cancer-free women. Significant weight gain was observed in survivors diagnosed less than 5 years prior to baseline (β = 3.81 pounds; 95% CI, 1.22–6.29) and women with estrogen receptor (ER)-negative tumors (β = 7.26 pounds; 95% CI, 2.23–12.30). Furthermore, survivors treated with chemotherapy were 2.1 times more likely to gain at least 11 pounds during follow-up compared with cancer-free women (OR, 2.10; 95% CI, 1.21–3.63). Weight gain was even greater among survivors who took statins while undergoing chemotherapy treatment (Pinteraction = 0.01). Conclusion: This is the first study to demonstrate that weight gain is an important issue in breast cancer survivors with a familial risk. In the first five years posttreatment, breast cancer survivors gain weight at a faster rate than cancer-free women, particularly after chemotherapy and statin use but not after hormone therapy alone. Impact: Our findings provide support for the development of weight gain interventions for young breast cancer survivors with a familial risk. Cancer Epidemiol Biomarkers Prev; 24(8); 1262–9. ©2015 AACR.

The proteasome ubiquitin receptor ADRM1 has been shown to be a driver for 20q13.3 amplification in epithelial cancers including ovarian and colon cancer. We performed array-CGH on 16 gastric cancer cell lines and found 20q13.3 to be amplified in 19% with the minimal amplified region in gastric cancer cell line AGS spanning a 1 Mb region including ADRM1. Expression microarray analysis shows overexpression of only two genes in the minimal region, ADRM1 and OSBPL2. While RNAi knockdown of both ADRM1 and OSBPL2 led to a slight reduction in growth, only ADRM1 RNAi knockdown led to a significant reduction in migration and growth in soft-agar. Treatment of AGS cells with the ADRM1 inhibitor RA190 resulted in proteasome inhibition, but RNAi knockdown of ADRM1 did not. However, RNAi knockdown of ADRM1 led to a significant reduction in specific proteins including MNAT1, HRS, and EGFR. We hypothesize that ADRM1 may act in ADRM1-amplified gastric cancer to alter protein levels of specific oncogenes resulting in an increase in metastatic potential. Selective inhibition of ADRM1 independent of proteasome inhibition may result in a targeted therapy for ADRM1-amplified gastric cancer. In vivo models are now warranted to validate these findings. © 2015 Wiley Periodicals, Inc.

Immunohistochemical expression of GATA-3 is seen predominantly in non-neoplastic bladder and breast epithelium and their respective carcinomas; however, data on expression in normal and lesional trophoblastic tissues are limited. Immunohistochemical staining for GATA-3 was assessed in a range of normal/lesional trophoblastic tissues and tumors in the differential diagnosis (n=445), including nonmolar products of conceptions/second and third trimester placentas/ectopic pregnancies, hydatidiform moles, placental site nodules, normal/exaggerated implantation sites, choriocarcinomas, epithelioid trophoblastic tumors, placental site trophoblastic tumors, atypical smooth muscle tumors (including leiomyosarcoma), and cervical and pulmonary squamous cell carcinomas. The extent of expression (0 to 4+) and intensity (weak to strong) were recorded. All cases with developing trophoblast/non-neoplastic trophoblastic proliferation and 81% of trophoblastic neoplasms were positive. Of all non-neoplastic trophoblast cell types, expression was observed in cytotrophoblast in 89% of cases, syncytiotrophoblast in 50%, intermediate trophoblast in 100%, and villous trophoblastic columns in 100%. Increasing gestational age was associated with a decrease in extent/intensity of expression in non-neoplastic cytotrophoblast and syncytiotrophoblast, whereas intermediate trophoblast maintained diffuse and strong expression from early to late gestation (P

Background Ovarian carcinoma is highly dependent on the ubiquitin proteasome system (UPS), but its clinical response to treatment with the proteasome inhibitor bortezomib has been disappointing. This has driven exploration of alternate approaches to target the UPS in ovarian cancer. Recently, proteasome inhibitors targeting the 19S regulatory particle-associated RPN13 protein have been described, such as RA190. RPN13, which is encoded by ADRM1, facilitates the recognition by the proteasome of its polyubiquinated substrates. Inhibition of RPN13 produces a rapid, toxic accumulation of polyubiquitinated proteins in ovarian and other cancer cells, triggering apoptosis. Here, we sought to determine if RPN13 is available as a target in precursors of ovarian/fallopian tube cancer as well as all advanced cases, and the impact of increased ADRM1 gene copy number on sensitivity of ovarian cancer to RA190. Methods ADRM1 mRNA was quantified by RNAscope in situ hybridization and RPN13 protein detected by immunohistochemistry in high grade serous carcinoma (HGSC) of the ovary and serous tubal intraepithelial carcinoma (STIC). Amplification of ADRM1 and sensitivity to RA190 were determined in ovarian cancer cell lines. Results Here, we demonstrate that expression of ADRM1mRNA is significantly elevated in STIC and HGSC as compared to normal fallopian tube epithelium. ADRM1 mRNA and RPN13 were ubiquitously and robustly expressed in ovarian carcinoma tissue and cell lines. No correlation was found between ADRM1 amplification and sensitivity of ovarian cancer cell lines to RA190, but all were susceptible. Conclusions RPN13 can potentially be targeted by RA190 in both in situ and metastatic ovarian carcinoma. Ovarian cancer cell lines are sensitive to RA190 regardless of whether the ADRM1 gene is amplified. Electronic supplementary material The online version of this article (doi:10.1186/s13048-017-0347-y) contains supplementary material, which is available to authorized users.

Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (∼20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (∼15%); and (iii) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8 + T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease. IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.

Aim To achieve durable and broad protection against human papillomaviruses by vaccination with multimers of minor capsid antigen L2 using self-adjuvanting fusions with the toll-like receptor-5 (TLR5) ligand bacterial flagellin (Fla) instead of co-formulation with alum. Methods Fla fusions with L2 protective epitopes comprising residues 11-200, 11-88 and/or 17-38 of a single or multiple HPV types were produced in E. coli and their capacity to activate TLR5 signaling was assessed. Immunogenicity was evaluated serially following administration of 3 intramuscular doses of Fla-L2 multimer without exogenous adjuvant, followed by challenge 1, 3, 6 or 12 months later, and efficacy compared to vaccination with human doses of L1 VLP vaccines (Gardasil and Cervarix) or L2 multimer formulated in alum. Serum antibody responses were assessed by peptide ELISA, in vitro neutralization assays and passive transfer to naive rabbits in which End-Point Protection Titers (EPPT) were determined using serial dilutions of pooled immune sera collected 1, 3, 6 or 12 months after completing active immunization. Efficacy was assessed by determining wart volume following concurrent challenge at different sites with HPV6/16/18/31/45/58 ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes. Results Vaccination in the absence of exogenous adjuvant with Fla-HPV16 L2 11-200 fusion protein elicited durable protection against HPV16, but limited cross-protection against other HPV types. Peptide mapping data suggested the importance of the 17-38 aa region in conferring immunity. Indeed, addition of L2 residues 17-38 of HPV6/18/31/39/52 to a Fla-HPV16 L2 11-200 or 11-88 elicited broader protection via active or passive immunization, similar to that seen with vaccination with an alum-adjuvanted L2 multimer comprising the aa 11-88 peptides of five or eight genital HPV types. Conclusions Vaccination with flagellin fused L2 multimers provided lasting (>1 year) immunity without the need for an exogenous adjuvant. Inclusion of the L2 amino acid 17-38 region in such multi-HPV type fusions expanded the spectrum of protection.

Purpose: Ovarian cancer is the deadliest of the gynecologic malignancies. Carcinogenic progression is accompanied by upregulation of ubiquitin-dependent protein degradation machinery as a mechanism to compensate with elevated endogenous proteotoxic stress. Recent studies support the notion that deubiquitinating enzymes (DUB) are essential factors in proteolytic degradation and that their aberrant activity is linked to cancer progression and chemoresistance. Thus, DUBs are an attractive therapeutic target for ovarian cancer. Experimental Design: The potency and selectivity of RA-9 inhibitor for proteasome-associated DUBs was determined in ovarian cancer cell lines and primary cells. The anticancer activity of RA-9 and its mechanism of action were evaluated in multiple cancer cell lines in vitro and in vivo in immunodeficient mice bearing an intraperitoneal ES-2 xenograft model of human ovarian cancer. Results: Here, we report the characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs. Treatment with RA-9 selectively induces onset of apoptosis in ovarian cancer cell lines and primary cultures derived from donors. Loss of cell viability following RA-9 exposure is associated with an unfolded protein response as mechanism to compensate for unsustainable levels of proteotoxic stress. In vivo treatment with RA-9 retards tumor growth, increases overall survival, and was well tolerated by the host. Conclusions: Our preclinical studies support further evaluation of RA-9 as an ovarian cancer therapeutic. Clin Cancer Res; 20(12); 3174–86. ©2014 AACR.

Genetically modified bacterial flagellin (Fla), a Toll-like receptor-5 (TLR5) ligand, was evaluated as a fusion partner for human papillomavirus (HPV) L2-based immunogens in two animal challenge models; either cutaneous inoculation of rabbits with HPV ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes that induce warts, or intra-vaginal inoculation of mice with HPV ‘pseudovirions’ encapsidating a luciferase reporter plasmid and measurement of bioluminescence to determine infectivity. An Escherichia coli production system was developed for flagellin-L2 (Fla-L2) fusions containing either monomeric HPV-16 L2 a.a. 11( × 11–200) or oligomeric L2 comprising a fusion of the a.a. 11–88 peptides of five (Fla∼5 × 11–88) or eight (Fla∼8 × 11–88) genital HPV types. Immunogenicity and bioactivity of Fla-L2 constructs were assessed using an in vitro neutralization and cell-based TLR-5 binding assay, respectively. Efficacy was evaluated following active immunization of rabbits or mice administered 3 intramuscular doses of Fla-L2 recombinants without exogenous adjuvant, followed by challenge. In addition, passive immunization studies of naive rabbits with serial dilutions of pooled immune sera were used to determine End-Point Protection Titers (EPPT) for each formulation against a broader spectrum of HPV quasivirions. Efficacy was assessed for up to 10 weeks on the basis of wart volume induced following challenge and results compared to licensed L1-VLP vaccines (Gardasil and Cervarix). Following active immunization at doses as low as 1 μg, Fla-L2 fusions afforded complete protection against infection (mice) and disease (rabbits) following either homologous or heterologous HPV challenge. Passive immunization with anti-L2 immune sera discriminated between the different vaccine candidates under evaluation, demonstrated the protective role of antibody and suggested the superiority of this oligomeric L2-TLR5 agonist fusion approach compared to L1-based vaccines in its ability to cross-protect against non-vaccine HPV types.

Purpose: Radiotherapy kills cancer cells by causing DNA damage, and stimulates a systemic antitumor immune response by releasing tumor antigen and endogenous adjuvant within the tumor microenvironment. However, radiotherapy also induces the recruitment of immunosuppressive myeloid cells, which can interfere with the antitumor immune responses elicited by apoptotic tumor cells. We hypothesized that local delivery of vaccine following radiotherapy will lead to the priming of antigen-specific CTL immune responses and render immunosuppressive myeloid cells susceptible to killing by the activated CTLs. Experimental Design: Using several antigenic systems, we tested whether intratumoral injection of antigenic peptide/protein in irradiated tumors would be able to prime CTLs as well as load myeloid cells with antigen, rendering them susceptible to antigen-specific CTL killing. Results: We show that by combining radiotherapy and targeted antigenic peptide delivery to the tumor, the adjuvant effect generated by radiotherapy itself was sufficient to elicit the priming and expansion of antigen-specific CTLs, through the type I IFN-dependent pathway, leading to synergistic therapeutic antitumor effects compared with either treatment alone. In addition, using two different types of transgenic mice, we demonstrated that CTL-mediated killing of stromal cells in tumors by our approach is important for tumor control. Finally, we confirmed the efficacy of this approach in our preclinical model using two clinically tested therapeutic human papilloma virus (HPV) vaccines. Conclusions: These data serve as an important foundation for the future clinical translation of radiotherapy combined with a clinically tested therapeutic HPV vaccine for the control of HPV-associated cancers. Clin Cancer Res; 20(3); 644–57. ©2013 AACR.

We show that minor capsid protein L2 is full length in clinical virion isolates and prepare furin-cleaved pseudovirus (fcPsV) as a model of the infectious intermediate for multiple human papillomavirus (HPV) types. These fcPsV do not require furin for in vitro infection, and are fully infectious in vivo. Both the γ-secretase inhibitor XXI and carrageenan block fcPsV infection in vitro and in vivo implying that they act after furin-cleavage of L2. Despite their enhanced exposure of L2 epitopes, vaccination with fcPsV particles fails to induce L2 antibody, although L1-specific responses are similar to PsV with intact L2. FcPsV can be applied in a simple, high-throughput neutralization assay that detects L2-specific neutralizing antibodies with >10-fold enhanced sensitivity compared with the PsV-based assay. The PsV and fcPsV-based assays exhibit similar sensitivity for type-specific antibodies elicited by L1 virus-like particles (VLP), but the latter improves detection of L1-specific cross-type neutralizing antibodies.

Background: The National Cancer Institute (NCI) PREVENT Cancer Program (PREVENT) is a peer-reviewed R&D pipeline with the core emphasis on preclinical development and clinical translation of novel cancer preventive interventions. One of the latest PREVENT-supported projects include cGMP production and IND enabling studies of a broad spectrum experimental human papillomavirus (HPV) vaccine- HPV16 RG1-VLP. This monovalent chimeric virus-like particle (VLP) displays 360 copies of the highly conserved epitope RG1 (aa 17-36 of minor capsid protein HPV16 L2) in the DE loop of HPV16L1 VLP backbone, and is capable of eliciting broadly neutralizing antibodies that target several clinically relevant HPV genotypes. RG-1 induced cross-neutralizing titers are typically lower than anti-L1 antibodies generated by currently licensed HPV vaccines. Hence, durability of protection has been cited as a cause of concern. Here, using engineering-run cGMP grade HPV16-RG1VLPs formulated with alhdyrogel®, the durability of protection 6 month post-vaccination of HPV16 RG1-VLPs against Gardasil-9®, a licensed HPV vaccine was evaluated using an established papillomavirus disease model. Methods: New Zealand white rabbits (n=15 per treatment group) were administered three intra-muscular vaccinations of HPV16-RG1 (80 µg), human doses of Gardasil-9®, or no vaccine (adjuvant only). Following vaccination, in vivo protection was assessed against 8 high-risk oncogenic HPVs utilizing methods from an established cottontail rabbit papillomavirus (CRPV) disease model. Within each treatment group, 5 rabbits were challenged two weeks post-final vaccination (at peak serum ELISA titer), while another 5 were challenged six months post-final vaccination to assess durability of protection. The remainder 5 rabbits will be challenged one year post final vaccination. Results: During the peak-titer period, rabbits vaccinated with monovalent HPV16-RG1VLP were protected from disease development, which was comparable to the protection afforded by Gardasil-9®. Six months after final vaccination, despite lower serum titers, HPV16-RG1VLP immunized rabbits were still protected from disease development with vaccine efficacy comparable to that of Gardasil-9®. And, in some instances, HPV16-RG1VLP vaccine demonstrated a superior cross-protection. Conclusions: Even as a monovalent formulation, HPV16 RG1 VLP vaccination was able to provide comparable protection against a number of high-risk oncogenic HPV types, including types not covered by Gardasil-9®, even after six months post-vaccination. As a monovalent VLP, HPV16 RG1 VLP holds promise as a broad-spectrum preventative vaccine candidate that could potentially provide broader protection at lower production costs. Studies evaluating protection one-year-post vaccination is currently in progress. Citation Format: Jiafen Hu, Karla Balogh, Ken Matsui, Huimin Tan, Pola Olczak, George Buchman, Brian Howard, Jonathan White, Michelle Kennedy, Shizuko Sei, Elizabeth Glaze, Sarah Brendle, Christina Schellenbacher, Reinhard Kirnbauer, Richard Roden, Robert Shoemaker, Neil Christensen, Joshua Weiyuan Wang. A cGMP-grade chimeric papillomavirus candidate vaccine (HPV16 RG1-VLP) confers long term cross-protection compared to a nonavalent hpv vaccine in a pre-clinical papillomavirus animal model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-200.

The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2.

Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLPs) self-assembled from major capsid protein L1, afford type-restricted protection against HPV types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical cancers (CxCas) and 90% of genital warts. However, they do not protect against less prevalent high-risk (HR) types causing 30% of CxCa, or cutaneous HPV. In contrast, vaccination with the minor capsid protein L2 induces low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16 L2 amino acids 17–36 (RG1 epitope) within the DE-surface loop of HPV16 L1 induced cross-neutralizing antisera. We hypothesized that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL (aluminum hydroxide plus 3-O-desacyl-4′-monophosphoryl lipid A) induced robust L2 antibodies (ELISA titers 2,500–12,500), which (cross-)neutralized mucosal HR HPV16/18/45/37/33/52/58/35/39/51/59/68/73/26/69/34/70, low-risk HPV6/11/32/40, and cutaneous HPV2/27/3/76 (titers 25–1,000) using native virion- or pseudovirion (PsV)-based assays, and a vigorous cytotoxic T lymphocyte response by enzyme-linked immunospot. In vivo, mice were efficiently protected against experimental vaginal challenge with mucosal HR PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.

Multiple classes of pharmacologic agents have the potential to induce the expression and release of proinflammatory factors from dying tumor cells. As a result, these cells can in theory elicit an immune response through various defined mechanisms to permanently eradicate disseminated cancer. However, the impact of chemotherapy on the tumor-specific immune response in the context of the tumor microenvironment is largely unknown. Within the tumor microenvironment, the immune response promoted by chemotherapy is antagonized by an immune-suppressive milieu, and the balance of these opposing forces dictates the clinical course of disease. Here, we report that high antigen exposure within the tumor microenvironment following chemotherapy is sufficient to skew this balance in favor of a productive immune response. In elevating antigen exposure, chemotherapy can achieve long-term control of tumor progression without the need of an additional adjuvant. We found that chemotherapy initiated this phenomenon in the tumor microenvironment through an accumulation of dendritic cells, which stimulated CD8+ T cells and the type I IFN pathway. From this conceptual base, we developed a simple approach to cancer therapy combining chemotherapy and vaccination that may be widely applicable. Cancer Res; 73(8); 2493–504. ©2013 AACR.

Purpose: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells. Experimental Design: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)–approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. Clin Cancer Res; 19(5); 1139–46. ©2012 AACR.

As compared with peptide- or protein-based vaccines, naked DNA vectors and even traditional attenuated or inactivated virus vaccines, virus-like particles (VLPs) are an attractive vaccine platform, as they offer a combination of safety, ease of production and both high-density B-cell epitope display and intracellular presentation of T-cell epitopes that induce potent humoral and cellular immune responses, respectively. Indeed, HPV vaccines based on VLP production by recombinant expression of major capsid antigen L1 in yeast (Gardasil(®), MerckCo., NJ, USA) or insect cells (Cervarix(®), GlaxoSmithKline, London, UK) have been licensed for the prevention of cervical and anogenital infection and disease associated with the genotypes targeted by each vaccine. However, these HPV vaccines have not been demonstrated as effective to treat existing infections, and efforts to develop a therapeutic HPV vaccine continue. Furthermore, current HPV L1-VLP vaccines provide type-restricted protection, requiring highly multivalent formulations to broaden coverage to the dozen or more oncogenic HPV genotypes. This raises the complexity and cost of vaccine production. The lack of access to screening and high disease burden in developing countries has spurred efforts to develop second-generation HPV vaccines that are more affordable, induce wider protective coverage and offer therapeutic coverage against HPV-associated malignancies. Given the previous success with L1-VLP-based vaccines against HPV, VLPs have been also adopted as platforms for many second-generation HPV and non-HPV vaccine candidates with both prophylactic and therapeutic intent. In this article, the authors examine the progress and challenges of these efforts, with a focus on how they inform VLP vaccine design.

Infections with sexually transmitted high-risk Human Papillomavirus (hrHPV), of which there are at least 15 genotypes, are responsible for a tremendous disease burden by causing cervical, and subsets of other ano-genital and oro-pharyngeal carcinomas, together representing 5% of all cancer cases worldwide. HPV subunit vaccines consisting of virus-like particles (VLP) self-assembled from major capsid protein L1 plus adjuvant have been licensed. Prophylactic vaccinations with the 2-valent (HPV16/18), 4-valent (HPV6/11 /16/18), or 9-valent (HPV6/11/16/18/31/33/45/52/58) vaccine induce high-titer neutralizing antibodies restricted to the vaccine types that cause up to 90% of cervical carcinomas, a subset of other ano-genital and oro-pharyngeal cancers and 90% of benign ano-genital warts (condylomata). The complexity of manufacturing multivalent L1-VLP vaccines limits the number of included VLP types and thus the vaccines’ spectrum of protection, leaving a panel of oncogenic mucosal HPV unaddressed. In addition, current vaccines do not protect against cutaneous HPV types causing benign skin warts, or against beta-papillomavirus (betaPV) types implicated in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. In contrast with L1-VLP, the minor capsid protein L2 contains type-common epitopes that induce low-titer yet broadly cross-neutralizing antibodies to heterologous PV types and provide cross-protection in animal challenge models. Efforts to increase the low immunogenicity of L2 (poly)-peptides and thereby to develop broader-spectrum HPV vaccines are the focus of this review.

Background Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer as well as subsets of anogenital and oropharyngeal cancers. The two HPV viral oncoproteins, E6 and E7, are uniquely and consistently expressed in all HPV infected cells and are therefore promising targets for therapeutic vaccination. Both recombinant naked DNA and protein-based HPV vaccines have been demonstrated to elicit HPV-specific CD8+ T cell responses that provide therapeutic effects against HPV-associated tumor models. Here we examine the immunogenicity in a preclinical model of priming with HPV DNA vaccine followed by boosting with filterable aggregates of HPV 16 L2E6E7 fusion protein (TA-CIN). Results We observed that priming twice with an HPV DNA vaccine followed by a single TA-CIN booster immunization generated the strongest antigen-specific CD8+ T cell response compared to other prime-boost combinations tested in C57BL/6 mice, whether naïve or bearing the HPV16 E6/E7 transformed syngeneic tumor model, TC-1. We showed that the magnitude of antigen-specific CD8+ T cell response generated by the DNA vaccine prime, TA-CIN protein vaccine boost combinatorial strategy is dependent on the dose of TA-CIN protein vaccine. In addition, we found that a single booster immunization comprising intradermal or intramuscular administration of TA-CIN after priming twice with an HPV DNA vaccine generated a comparable boost to E7-specific CD8+ T cell responses. We also demonstrated that the immune responses elicited by the DNA vaccine prime, TA-CIN protein vaccine boost strategy translate into potent prophylactic and therapeutic antitumor effects. Finally, as seen for repeat TA-CIN protein vaccination, we showed that the heterologous DNA prime and protein boost vaccination strategy is well tolerated by mice. Conclusions Our results provide rationale for future clinical testing of HPV DNA vaccine prime, TA-CIN protein vaccine boost immunization regimen for the control of HPV-associated diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13578-016-0080-z) contains supplementary material, which is available to authorized users.

Recently, we reported that bisbenzylidine piperidone RA190 adducts to Cys-88 of the proteasome ubiquitin receptor hRpn13, triggering accumulation of ubiquitinated proteins and endoplasmic reticulum stress-related apoptosis in various cancer cell lines. hRpn13 contains an N-terminal pleckstrin-like receptor for ubiquitin domain that binds ubiquitin and docks it into the proteasome as well as a C-terminal deubiquitinase adaptor (DEUBAD) domain that binds the deubiquitinating enzyme Uch37. Here we report that hRpn13 and Uch37 are required for proper cell cycle progression and that their protein knockdown leads to stalling at G0/G1. Moreover, serum-starved cells display reduced hRpn13 and Uch37 protein levels with hallmarks of G0/G1 stalling and recovery to their steady-state protein levels following release from nutrient deprivation. Interestingly, loss of hRpn13 correlates with a small but statistically significant reduction in Uch37 protein levels, suggesting that hRpn13 interaction may stabilize this deubiquitinating enzyme in human cells. We also find that RA190 treatment leads to a loss of S phase, suggesting a block of DNA replication, and G2 arrest by using fluorescence-activated cell sorting. Uch37 deprivation further indicated a reduction of DNA replication and G0/G1 stalling. Overall, this work implicates hRpn13 and Uch37 in cell cycle progression, providing a rationale for their function in cellular proliferation and for the apoptotic effect of the hRpn13-targeting molecule RA190.

// Ruey-Shyang Soong 1, 5, 6 , Ravi K. Anchoori 4 , Benjamin Yang 1 , Andrew Yang 1 , Ssu-Hsueh Tseng 1 , Liangmei He 1 , Ya-Chea Tsai 1 , Richard B.S. Roden 1, 2, 4 , Chien-Fu Hung 1, 4 1 Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 2 Department of Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 3 Department of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 4 Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 5 Department of General Surgery, Chang Gung Memorial Hospital at Keelung, Keelung City, Taiwan 6 Department of Chang Gung University, College of Medicine, Taoyuan, Taiwan Correspondence to: Chien-Fu Hung, email: chung2@jhmi.edu Keywords: RPN13, proteasome, immunosuppression, MDSCs, Stat3 Received: January 14, 2016 Accepted: September 12, 2016 Published: September 17, 2016 ABSTRACT Myeloid-derived-suppressor cells (MDSCs) are key mediators of immune suppression in the ovarian tumor microenvironment. Modulation of MDSC function to relieve immunosuppression may enhance the immunologic clearance of tumors. The bis-benzylidine piperidone RA190 binds to the ubiquitin receptor RPN13/ADRM1 on the 19S regulatory particle of the proteasome and directly kills ovarian cancer cells by triggering proteotoxic stress. Here we examine the effect of RA190 treatment on the immunosuppression induced by MDSCs in the tumor microenvironment, specifically on the immunosuppression induced by MDSCs. We show that RA190 reduces the expression of Stat3 and the levels of key immunosuppressive enzymes and cytokines arginase, iNOS, and IL-10 in MDSCs, while boosting expression of the immunostimulatory cytokine IL-12. Furthermore, we show that the RA190-treated MDSCs lost their capacity to suppress CD8+ T cell function. Finally, we show that RA190 treatment of mice bearing syngeneic ovarian tumor elicits potent CD8+ T cell antitumor immune responses and improves tumor control and survival. These data suggest the potential of RA190 for ovarian cancer treatment by both direct killing of tumor cells via proteasome inhibition and relief of MDSC-mediated suppression of CD8 T cell-dependent antitumor immunity elicited by the apoptotic tumor cells.

In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein—L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal (462LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.

Ovarian cancer is the most lethal gynecologic malignancy in adult women. The origin of epithelial ovarian tumors is both morphologically and biologically heterogeneous, and different subtypes of ovarian tumors have different clinical outcomes. In spite of the heterogeneous nature of ovarian carcinoma, the current biomarkers and treatments for this disease are not subtype-specific. To discover the molecular basis of the ovarian tumor subtypes, we analyzed extracellular glycoproteins of seven common subtypes and normal ovary tissues using quantitative glycoproteomic analysis. Glycoproteins for different ovarian tumor subtypes were identified by liquid chromatography-tandem mass spectrometry and quantitated by spectral counting and then verified by iTRAQ labeling and Western blotting. Glycoproteins uniquely expressed in different subtypes of ovarian tumors or commonly expressed in most subtypes were identified. Using Western blots, we verified that mesothelin was overexpressed in serous carcinoma and transitional-cell carcinoma, CEA5 and CEA6 were overexpressed only in mucinous carcinoma, while versican and periostin were overexpressed in most subtypes of ovarian tumors. This study represents the first proteomic characterization of different ovarian tumor subtypes. The identified glycoproteins for histological subtypes of ovarian tumors will facilitate the understanding of the molecular basis, diagnosis of ovarian tumor subtypes, and predictions for treatment responses to therapeutic agents.

It is unclear what level of neutralizing antibody is sufficient to protect cattle from experimental bovine papillomavirus type 4 (BPV4) challenge. Markedly lower, and often undetected, serum neutralizing antibody titers were associated with protection in cattle vaccinated with BPV4 L2 as compared to L1 VLP. We hypothesized that vaccination with concatemers of the N-terminal protective epitopes of L2 derived from multiple animal papillomavirus types would enhance the breadth and strength of immunity. Therefore we generated a multimeric L2 antigen derived from three bovine and three canine papillomavirus types with divergent phenotypes and purified it from bacteria. Mice vaccinated three times with this six type L2 vaccine formulated in alum or RIBI adjuvant generated robust serum neutralizing antibody titers against BPV1, BPV4 and canine oral papillomavirus (COPV). Furthermore, vaccination with this six type L2 vaccine formulated in adjuvant, like BPV1 L1 VLP, protected the mice from experimental challenge with BPV1 pseudovirus.

Approximately 10% to 15% of human cancers lack detectable telomerase activity, and a subset of these maintain telomere lengths by the telomerase-independent telomere maintenance mechanism termed alternative lengthening of telomeres (ALT). The ALT phenotype, relatively common in subtypes of sarcomas and astrocytomas, has rarely been reported in epithelial malignancies. However, the prevalence of ALT has not been thoroughly assessed across all cancer types. We therefore comprehensively surveyed the ALT phenotype in a broad range of human cancers. In total, two independent sets comprising 6110 primary tumors from 94 different cancer subtypes, 541 benign neoplasms, and 264 normal tissue samples were assessed by combined telomere-specific fluorescence in situ hybridization and immunofluorescence labeling for PML protein. Overall, ALT was observed in 3.73% (228/6110) of all tumor specimens, but was not observed in benign neoplasms or normal tissues. This is the first report of ALT in carcinomas arising from the bladder, cervix, endometrium, esophagus, gallbladder, kidney, liver, and lung. Additionally, this is the first report of ALT in medulloblastomas, oligodendrogliomas, meningiomas, schwannomas, and pediatric glioblastoma multiformes. Previous studies have shown associations between ALT status and prognosis in some tumor types; thus, further studies are warranted to assess the potential prognostic significance and unique biology of ALT-positive tumors. These findings may have therapeutic consequences, because ALT-positive cancers are predicted to be resistant to anti-telomerase therapies.

Oncogenic human papillomaviruses (HPVs) are exclusively mucosal pathogens that are noncytopathic and the basal epithelial cells harboring and maintaining an infection do not produce either capsid antigen or virus. The efficacy of the licensed L1 virus-like particle (VLP) vaccines has encouraged development of several second generation vaccines aimed at expanding the coverage to all oncogenic HPV types and reducing barriers to global implementation. Currently there is no defined immune correlate of protection that can be used to determine if an individual patient is protected and for the evaluation of these second generation vaccines. Surprisingly, passive transfer of neutralizing serum antibody is protective in animal models. Recent studies suggest how neutralizing antibody mediates immunity against mucosal HPV and the possible impact of memory B cells.

Remodeling Gone Awry The identification of genes that are mutated at high frequency in human tumors can provide important clues to the molecular pathways that drive tumor growth, which in turn can potentially lead to more effective therapies. Jones et al. (p. 228 , published online 9 September; see the cover) looked for such mutations in ovarian clear cell carcinoma, a rare but particularly lethal form of ovarian cancer. Of 42 tumors examined, 57% were found to harbor inactivating mutations in ARID1A , a gene coding for a subunit of the SWI/SNF chromatin remodeling complex, which functions as a master regulator of transcription factor action and gene expression. Thus, proteins associated with the epigenetic control of gene expression can contribute to the development of human cancer.

SummaryUsing a human papillomavirus (HPV) cervicovaginal murine challenge model, we microscopically examined the in vivo mechanisms of L1 virus-like particle (VLP) and L2 vaccine-induced inhibition of infection. In vivo HPV infection requires an initial association with the acellular basement membrane (BM) to induce conformational changes in the virion that permit its association with the keratinocyte cell surface. By passive transfer of immune serum, we determined that anti-L1 antibodies can interfere with infection at two stages. Similarly to active VLP immunization, transfer of high L1 antibody concentrations prevented BM binding. However, in the presence of low concentrations of anti-L1, virions associated with the BM, but to the epithelial cell surface was not detected. Regardless of the concentration, L2 vaccine-induced antibodies allow BM association but prevent association with the cell surface. Thus, we have revealed distinct mechanisms of vaccine-induced inhibition of virus infection in vivo.

Background: Vulval intraepithelial neoplasia (VIN) is a premalignant condition, which is frequently associated with type HPV16 infection, and multifocal disease has high rates of surgical treatment failure. Methods: We report a phase II clinical trial of the topical immunomodulator, imiquimod, for 8 weeks, followed by 3 doses (weeks 10, 14 and 18) of therapeutic human papillomavirus (HPV) vaccination (TA-CIN, fusion protein HPV16 E6E7L2) in 19 women with VIN grades 2 and 3. Histology and HPV testing of biopsies were performed at weeks 0, 10, 20 and 52. Intralesional infiltration of T-cell subsets and lymphocyte proliferation for HPV systemic immune responses were also assessed. Results: Lesion response (complete regression of VIN on histology) was observed in 32% (6 out of 19) of women at week 10, increasing to 58% (11 out of 19) at week 20 and 63% (12 out of 19) at week 52. At this time, 36% (5 out of 14) of lesions showed HPV16 clearance and 79% (15 out of 19) of women were symptom free. At week 20, after treatment with imiquimod and vaccination, there was significantly increased local infiltration of CD8 and CD4 T cells in lesion responders; in contrast, non-responders (persistent VIN by histology) showed an increased density of T regulatory cells. After vaccination, only lesion responders had significantly increased lympho-proliferation to the HPV vaccine antigens. Conclusion: The therapeutic effect of treatment depends on the differential immune response of responders and non-responders with affect locally and systemically.

Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.

The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund's adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund's adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.

Background Vaccination with minor capsid protein L2 induces antibodies that cross-neutralize diverse papillomavirus types. However, neutralizing antibody titers against the papillomavirus type from which the L2 vaccine was derived are generally higher than the titers against heterologous types, which could limit effectiveness against heterologous types. We hypothesized that vaccination with concatenated multitype L2 fusion proteins derived from known cross-protective epitopes of several divergent human papillomavirus (HPV) types might enhance immunity across clinically relevant HPV genotypes. Methods Antibody responses of mice (n = 120) and rabbits (n = 23) to vaccination with HPV-16 amino-terminal L2 polypeptides or multitype L2 fusion proteins, namely, 11-200 × 3 (HPV types 6, 16, 18), 11-88 × 5 (HPV types 1, 5, 6, 16, 18), or 17-36 × 22 (five cutaneous, two mucosal low-risk, and 15 oncogenic types), that were formulated alone or in GPI-0100, alum, or 1018 ISS adjuvants were compared with vaccination with L1 virus-like particles (VLPs), including Gardasil, a licensed quadrivalent HPV L1 vaccine, and a negative control. Mice were challenged with HPV-16 pseudovirions 4 months after vaccination. Statistical tests were two-sided. Results The HPV-16 L2 polypeptides generated robust HPV-16–neutralizing antibody responses, albeit lower than those to HPV-16 L1 VLPs, and lower responses against other HPVs. In contrast, vaccination with the multitype L2 fusion proteins 11-200 x 3 and 11-88 x 5 induced high serum neutralizing antibody titers against all heterologous HPVs tested. 11-200 × 3 formulated in GPI-0100 adjuvant or alum with 1018 ISS protected mice against HPV-16 challenge (reduction in HPV-16 infection vs phosphate-buffered saline control, P < .001) 4 months after vaccination as well as HPV-16 L1 VLPs, but 11-200 × 3 alone or formulated with either alum or 1018 ISS was less effective (reduction in HPV-16 infection, P < .001). Conclusion Concatenated multitype L2 proteins in adjuvant have potential as pan-oncogenic HPV vaccines.

Purpose: Cervical cancer cells are addicted to the expression of the human papillomavirus (HPV) oncoproteins E6 and E7. The oncogencity of E6 is mediated in part by targeting p53 and PDZ-family tumor suppressor proteins for rapid proteasomal degradation, whereas the E7 oncoprotein acts in part by coopting histone deacetylases (HDAC)1/2. Here, we examine the hypothesis that inhibition of proteasome function and HDAC activity would synergistically and specifically trigger cervical cancer cell death by the interruption of E6 and E7 signaling. Experimental Design: The sensitivity and molecular responses of keratinocytes and HPV-positive and HPV-negative cervical cancer cells and xenografts to combinations of proteasome and HDAC inhibitors were tested. The expression of HDAC1/HDAC2 in situ was examined in cervical cancer, its precursors, and normal epithelium. Results: Cervical cancer cell lines exhibit greater sensitivity to proteasome inhibitors than do HPV-negative cervical cancers or primary human keratinocytes. Treatment of cervical cancer cells with bortezomib elevated the level of p53 but not hDlg, hScribble or hMAGI. Immunohistochemical analysis revealed elevated HDAC1/HDAC2 expression in cervical dysplasia and cervical carcinoma versus normal cervical epithelium. The combination of bortezomib and HDAC inhibitor trichostatin A or vorinostat shows synergistic killing of HPV-positive, but not HPV-negative, cervical cancer cell lines. Similarly, treatment of HeLa xenografts with the combination of bortezomib and trichostatin A retarded tumor growth significantly more effectively than either agent alone. Conclusions: A combination of proteasome and HDAC inhibitors, including bortezomib and vorinostat, respectively, warrants exploration for the treatment of cervical cancer.

The expression pattern of human papillomavirus (HPV) capsid antigen L2 is poorly described, and the significance of its localization with both promyelocytic leukemia protein (PML) and Daxx in a subnuclear domain, nuclear domain 10 (ND-10), when ectopically expressed in tissue culture cells is controversial. To address whether ND-10 localization of L2 occurs in natural cervical lesions, we used a HPV16 and HPV18 L2-specific monoclonal antibody (RG-1), in addition to rabbit antiserum to HPV6 L2, to localize L2. Immunohistochemical staining with RG-1 produced diffuse staining in the nuclei of some cells located within the superficial epithelial layers in eight of nine cases of HPV16/18(+) cervical intraepithelial neoplasia grade 1 (CIN1); however, no staining was observed in HPV16/18(+) high-grade CIN (0 of 8 cases), normal cervical epithelium (0 of 20 cases), cervical squamous cell carcinoma (0 of 102 cases), adenocarcinoma (0 of 51 cases), or adenosquamous carcinoma (0 of 6 cases). HPV16/18(+) cervical lesions that express L2 exhibit higher HPV16/18 genome copies per cell compared with those that do not positively stain with RG-1 (P = 0.04). RG-1 staining of HeLa cells transfected with L2 expression constructs was frequently concentrated in the ND-10, particularly in cells expressing high levels of L2, and co-localized with the cellular markers of ND-10, PML, and Daxx. In contrast, L2 was primarily diffuse within the nucleus and distinct from ND-10 as defined by PML immunofluorescent staining in CIN lesions, condylomata, and HPV16-transduced organotypic cultures.

Purpose: Elevated metabolic activity of ovarian cancer cells causes increased ubiquitin-proteasome-system (UPS) stress, resulting in their greater sensitivity to the toxic effects of proteasomal inhibition. The proteasomes and a potentially compensatory histone deacetylase 6 (HDAC6)-dependent lysosomal pathway mediate eukaryotic protein turnover. We hypothesized that up-regulation of the HDAC6-dependent lysosomal pathway occurs in response to UPS stress and proteasomal inhibition, and thus, ovarian cancer cell death can be triggered most effectively by coinhibition of both the proteasome- and HDAC6-dependent protein degradation pathways. Experimental Design: To address this hypothesis, we examined HDAC6 expression patterns in normal and cancerous ovarian tissues and used a novel HDAC6-specific inhibitor, NK84, to address HDAC6 function in ovarian cancer. Results: Abnormally high levels of HDAC6 are expressed by ovarian cancer cells in situ and in culture relative to benign epithelium and immortalized ovarian surface epithelium, respectively. Specific HDAC6 inhibition acts in synergy with the proteasome inhibitor Bortezomib (PS-341) to cause selective apoptotic cell death of ovarian cancer cells at doses that do not cause significant toxicity when used individually. Levels of UPS stress regulate the sensitivity of ovarian cancer cells to proteasome/HDAC6 inhibition. Pharmacologic inhibition of HDAC6 also reduces ovarian cancer cell spreading and migration consistent with its known function in regulating microtubule polymerization via deacetylation of α-tubulin. Conclusion: Our results suggest the elevation of both the proteasomal and alternate HDAC6-dependent proteolytic pathways in ovarian cancer and the potential of combined inhibition of proteasome and HDAC6 as a therapy for ovarian cancer.

Pseudovirions of human papillomavirus type 16 (HPV16), the principal etiologic agent in 50% of cervical cancers, were used as a model system to investigate the cell surface interactions involved in the exposure of the broadly cross-neutralizing papillomavirus L2 epitopes. These neutralizing epitopes were exposed only after cell surface binding and a subsequent change in capsid conformation that permitted cleavage by the cellular protease furin at a specific highly conserved site in L2 that is immediately upstream of the cross-neutralizing epitopes. Unexpectedly, binding of L2 antibodies led to the release of the capsid/antibody complexes from the cell surface and their accumulation on the extracellular matrix. Study of the dynamics of exposure of the L2 epitopes further revealed that representatives of the apparently dominant class of L1-specific neutralizing antibodies induced by virus-like particle vaccination prevent infection, not by preventing cell surface binding but rather by preventing the conformation change involved in exposure of the L2 neutralizing epitope. These findings suggest a dynamic model of virion-cell surface interactions that has implications for both evolution of viral serotypes and the efficacy of current and future HPV vaccines.

This perspective aims to look at how the recently licensed human papillomavirus (HPV) vaccines, based on L1 virus-like particles (VLPs), will impact cervical cancer globally, and what remains to be done in this field. The current VLP vaccines protect against infection from a substantial proportion, but not all, of the oncogenic HPV genotypes, and they have no proven therapeutic benefit for patients with HPV disease. Furthermore, they are too costly for sustained global use, and it is unclear how they may impact current cytologic screening protocols. A number of new technologies show promise in addressing these issues, including molecular screening tools and alternative vaccine strategies.

Cervical cancer continues to cause significant morbidity and mortality worldwide, making prophylactic cervical cancer vaccines an important focus for cervical cancer prevention. The increasing accessibility of these vaccines worldwide has the potential to greatly decrease the incidence and burden of disease in the future. However, current prophylactic vaccines offer no therapeutic benefit for persons already infected with human papillomavirus types targeted by vaccines or persons with precancerous lesions or cervical cancer. The protection offered by current vaccines is primarily against human papillomavirus types used to derive the vaccine, although partial cross-protection for related virus types has been observed. Herein, we describe findings from preclinical and clinical studies that employ vaccine strategies that have the potential to shape the future of vaccines against cervical cancer. Modalities include prophylactic strategies to target more oncogenic virus types by using the minor capsid antigen L2 and/or by increasing the number of types used to derive virus-like particle vaccines. Therapeutic strategies include the development of vaccines against human papillomavirus early proteins (targets for cellular immunity) for the resolution of precancerous lesions and cervical cancer. Future applications of existing VLP-based vaccines are also discussed.

We contrast the efforts to treat ovarian cancer and cervical cancer through vaccination because of their different pathobiology. A plethora of approaches have been developed for therapeutic vaccination against cancer, many of which target defined tumor-associated antigens (TAAs). Persistent infection with oncogenic human papillomavirus (HPV) types is necessary cause of cervical cancer. Furthermore, cervical cancer patients frequently mount both humoral and T cell immune responses to the HPV E6 and E7 oncoproteins, whose expression is required for the transformed phenotype. Numerous vaccine studies target these viral TAAs, including recent trials that may enhance clearance of pre-malignant disease. By contrast little is known about the etiology of epithelial ovarian cancer. Although it is clear that p53 mutation or loss is a critical early event in the development of epithelial ovarian cancer, no precursor lesion has been described for the most common serous histotype, and even the location of its origin is debated. These issues have complicated the selection of appropriate ovarian TAAs and the design of vaccines. Here we focus on mesothelin as a promising ovarian TAA because it is overexpressed and immunogenic at high frequency in patients, is displayed on the cell surface and potentially contributes to ovarian cancer biology.

Cervical cancer is one of the most common cancers in women worldwide. Persistent infection with human papillomavirus (HPV) is considered to be the etiological factor for cervical cancer. Therefore, an effective vaccine against HPV infections may lead to the control of cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infections as well as cell-mediated immunity to eliminate established infection or HPV-related disease. In the current study, we have generated a potential preventive and therapeutic HPV DNA vaccine using human calreticulin (CRT) linked to HPV16 early proteins, E6 and E7 and the late protein L2 (hCRTE6E7L2). We found that vaccination with hCRTE6E7L2 DNA vaccine induced a potent E6/E7-specific CD8+ T cell immune response, resulting in a significant therapeutic effect against E6/E7 expressing tumor cells. In addition, vaccination with hCRTE6E7L2 DNA generated significant L2-specific neutralizing antibody responses, protecting against pseudovirion infection. Thus, the hCRTE6E7L2 DNA vaccines are capable of generating potent preventive and therapeutic effects in vaccinated mice. Our data has significant clinical implications.

We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.

Both tumor cell proliferation and metastasis are dependent on myosin II. Because UNC-45 is required to chaperone the assembly of a functional myosin II motor, we examined the expression of the general cell (GC) UNC-45 isoform in ovarian tumors. Serous carcinoma expressed elevated levels of GC UNC-45 compared with normal ovarian surface epithelium and benign cystadenoma. High-stage exhibited greater GC UNC-45 expression than low-stage serous carcinoma. Similarly, GC UNC-45 transcripts and protein levels were higher in ovarian cell lines than in immortalized ovarian surface epithelial cells. Elevation of GC UNC-45 levels by ectopic expression enhanced the rate of ovarian cancer cell proliferation, whereas siRNA knockdown of GC UNC-45 suppressed proliferation without altering myosin II levels. GC UNC-45 and myosin II were diffuse within the cytoplasm of confluent interphase cells, but both accumulated together at the cleavage furrow during cytokinesis. GC UNC-45 and myosin II also trafficked to the leading edges of ovarian cancer cells induced to move in a scratch assay. Knockdown of GC UNC-45 reduced the spreading ability of ovarian cancer cells whereas it was enhanced by GC UNC-45 overexpression. In sum, these findings implicate elevated GC UNC-45 protein expression in ovarian carcinoma proliferation and metastasis.

Persistent papillomas developed in ~10% of out-bred immune-competent SKH-1 mice following MusPV1 challenge of their tail, and in a similar fraction the papillomas were transient, suggesting potential as a model. However, papillomas only occurred in BALB/c or C57BL/6 mice depleted of T cells with anti-CD3 antibody, and they completely regressed within 8 weeks after depletion was stopped. Neither CD4+ nor CD8+ T cell depletion alone in BALB/c or C57BL/6 mice was sufficient to permit visible papilloma formation. However, low levels of MusPV1 were sporadically detected by either genomic DNA-specific PCR analysis of local skin swabs or in situ hybridization of the challenge site with an E6/E7 probe. After switching to CD3+ T cell depletion, papillomas appeared upon 14/15 of mice that had been CD4+ T cell depleted throughout the challenge phase, 1/15 of CD8+ T cell depleted mice, and none in mice without any prior T cell depletion. Both control animals and those depleted with CD8-specific antibody generated MusPV1 L1 capsid-specific antibodies, but not those depleted with CD4-specific antibody prior to T cell depletion with CD3 antibody. Thus, normal BALB/c or C57BL/6 mice eliminate the challenge dose, whereas infection is suppressed but not completely cleared if their CD4 or CD8 T cells are depleted, and recrudescence of MusPV1 is much greater in the former following treatment with CD3 antibody, possibly reflecting their failure to generate capsid antibody. Systemic vaccination of C57BL/6 mice with DNA vectors expressing MusPV1 E6 or E7 fused to calreticulin elicits potent CD8 T cell responses and these immunodominant CD8 T cell epitopes were mapped. Adoptive transfer of a MusPV1 E6-specific CD8+ T cell line controlled established MusPV1 infection and papilloma in RAG1-knockout mice. These findings suggest the potential of immunotherapy for HPV-related disease and the importance of host immunogenetics in the outcome of infection., Author Summary While most patients clear human papillomavirus (HPV) infection, some develop persistent papillomas, especially if immunocompromised. Likewise, we find a fraction of outbred SKH-1 mice challenged with Mus musculus papillomavirus type 1 (MusPV1/MmuPV1) develop persistent papillomas, whereas most SKH-1 mice, as seen for the inbred C57BL/6 and BALB/c strains, clear the infection. Viral clearance requires both CD4+ and CD8+ T cells, and depletion of either subset permits persistent but subclinical infection. In C57BL/6 mice, CD8+ T cell epitopes were mapped to MusPV1 E6 and E7; however the CD8+ T cell response to E6 dominated and correlated with spontaneous regression. A MusPV1 E6-specific CD8+ T cell line was developed by vaccination and culture in vitro, and its systemic administration once was sufficient to effect papilloma clearance in an immunodeficient mouse. Our observations in inbred and outbred mice challenged with MusPV1 suggest promise for immunotherapy to treat HPV-associated disease.

High-grade epithelial ovarian cancer kills more women than any other gynecologic cancer and is rarely diagnosed at an early stage. We sought to identify tumor-associated antigens (TAA) as candidate diagnostic and/or immunotherapeutic targets by taking advantage of tumor autoantibody responses in individuals with ovarian cancer. Plasma-derived IgG from a pool of five patients with advanced ovarian cancer was subjected to iterative biopanning using a library of bacteriophage MS2 virus-like particles (MS2-VLPs) displaying diverse short random peptides. After two rounds of biopanning, we analyzed the selectant population of MS2-VLPs by Ion Torrent deep sequencing. One of the top 25 most abundant peptides identified (DISGTNTSRA) had sequence similarity to cancer antigen 125 (CA125/MUC16), a well-known ovarian cancer–associated antigen. Mice immunized with MS2-DISGTNTSRA generated antibodies that cross-reacted with purified soluble CA125 from ovarian cancer cells but not membrane-bound CA125, indicating that the DISGTNTSRA peptide was a CA125/MUC16 peptide mimic of soluble CA125. Preoperative ovarian cancer patient plasma (n = 100) was assessed for anti-DISGTNTSRA, anti-CA125, and CA125. Patients with normal CA125 (35 IU/mL). A statistically significant survival advantage was observed for patients who had either normal CA125 and/or higher concentrations of antibodies to CA125 at the time of diagnosis. These data show the feasibility of using deep sequence–coupled biopanning to identify TAA autoantibody responses from cancer patient plasma and suggest a possible antibody-mediated mechanism for low CA125 plasma concentrations in some ovarian cancer patients. Cancer Immunol Res; 4(2); 157–64. ©2015 AACR.

Background DNA vaccines have emerged as attractive candidates for the control of human papillomavirus (HPV)-associated malignancies. However, DNA vaccines suffer from limited immunogenicity and thus strategies to enhance DNA vaccine potency are needed. We have previously demonstrated that for DNA vaccines encoding HPV-16 E7 antigen (CRT/E7) linkage with calreticulin (CRT) linked enhances both the E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. In the current study, we aim to introduce an approach to elicit potent CD4+ T cell help for the enhancement of antigen-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination by using co-administration of a DNA vector expressing papillomavirus major and minor capsid antigens, L1 and L2. Result We showed that co-administration of vectors containing codon-optimized bovine papillomavirus type 1 (BPV-1) L1 and L2 in combination with DNA vaccines could elicit enhanced antigen-specific CD8+ in both CRT/E7 and ovalbumin (OVA) antigenic systems. We also demonstrated that co-administration of vectors expressing BPV-1 L1 and/or L2 DNA with CRT/E7 DNA led to the generation of L1/L2-specific CD4+ T cell immune responses and L1-specific neutralizing antibodies. Furthermore, we showed that co-administration with DNA encoding BPV1 L1 significantly enhances the therapeutic antitumor effects generated by CRT/E7 DNA vaccination. In addition, the observed enhancement of CD8+ T cell immune responses by DNA encoding L1 and L2 was also found to extend to HPV-16 L1/L2 system. Conclusion Our strategy elicits both potent neutralizing antibody and therapeutic responses and may potentially be extended to other antigenic systems beyond papillomavirus for the control of infection and/or cancer.

// Rachel Isaksson Vogel 1 , Kathleen Coughlin 2 , Alessandra Scotti 3 , Yoshie Iizuka 2 , Ravi Anchoori 4, 5 , Richard B. S. Roden 4, 5, 6 , Mauro Marastoni 3 , Martina Bazzaro 1, 2 1 Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA 2 Department of Obstetrics, Gynecology and Women's Heath, University of Minnesota, Minneapolis, Minnesota, USA 3 Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy 4 Department of Pathology, Johns Hopkins University, Baltimore, MD, USA 5 Department of Oncology, Johns Hopkins University, Baltimore, MD, USA 6 Department of Gynecology and Obstetrics, Johns Hopkins University, Baltimore, MD, USA Correspondence to: Martina Bazzaro, e-mail: mbazzaro@umn.edu Keywords: Breast Cancer, Triple Negative Breast Cancer, Proteasome Inhibitors, Deubiquitinating Enzymes, Autophagy Received: October 01, 2014 Accepted: December 11, 2014 Published: February 27, 2015 ABSTRACT Breast cancer is one of the leading causes of cancer death among women in the United States. Patients expressing the estrogen and progesterone receptor (ER and PR) and human epidermal growth factor 2 (HER-2) tumor markers have favorable prognosis and efficacious therapeutic options. In contrast, tumors that are negative for these markers (triple-negative) have a disproportionate share of morbidity and mortality due to lack of a validated molecular target. Deubiquitinating enzymes (DUBs) are a critical component of ubiquitin-proteasome-system degradation and have been shown to be differentially expressed and activated in a number of cancers, including breast, with their aberrant activity linked to cancer prognosis and clinical outcome. We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines. Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress. Treatment with sub-optimal doses of DUB and lysosome inhibitors synergistically kills TNBC cells. This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC.