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Current preclinical models for cervical cancer lack important clinical and pathologic features. To improve upon these models, we aimed to develop a novel, spontaneous HPV16-expressing carcinoma model that captures major aspects of HPV-associated cancer in the female genital tract. This novel preclinical model features (i) expression of HPV oncogenes E6 and E7 in the tumors in female reproductive tract of mice, (ii) spontaneous progression through high-grade squamous intraepithelial lesion (HSIL) to carcinoma, and (iii) flexibility to model cancers from different high-risk HPV genotypes. This was accomplished by injecting plasmids expressing HPV16 E6/E7-luciferase, AKT, c-myc, and Sleeping Beauty transposase into the cervicovaginal tract of C57BL/6 mice followed by electroporation. Cell lines derived from these tumors expressed HPV16 E6/E7 oncogenes, formed tumors in immunocompetent mice, and displayed carcinoma morphology. In all, this novel HPV-associated cervicogenital carcinoma model and HPV16E6/E7-expressing tumor cell line improves upon current HPV16-E6/E7-expressing tumor models. These tumor models may serve as important preclinical models for the development of therapeutic HPV vaccines or novel therapeutic interventions against HPV E6/E7-expressing tumors. SIGNIFICANCE: This study describes the development of a clinically relevant mouse model of cervicovaginal carcinoma that progresses from high-grade lesions and recapitulates key features of human HPV + cervical cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

Chronic inflammation induced by persistent microbial infection plays an essential role in tumor progression. Although it is well documented that Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC), how EBV-induced inflammation promotes NPC progression remains largely unknown. Here, we report that tumor infiltration of tumor-associated macrophages (TAM) and expression of CCL18, the cytokine preferentially secreted by TAM, closely correlate with serum EBV infection titers and tumor progression in two cohorts of NPC patients. In vitro, compared with EBV - NPC cell lines, EBV + NPC cell lines exhibited superior capacity to attract monocytes and skew them to differentiate to a TAM-like phenotype. Cytokine profiling analysis revealed that NPC cells with active EBV replications recruited monocytes by VEGF and induced TAM by GM-CSF in an NF-κB-dependent manner. Reciprocally, TAM induced epithelial-mesenchymal transition and furthered NF-κB activation of tumor cells by CCL18. In humanized mice, NPC cells with active EBV replications exhibited increased metastasis, and neutralization of CCL18, GM-CSF, and VEGF significantly reduced metastasis. Collectively, our work defines a feed-forward loop between tumor cells and macrophages in NPC, which shows how metastatic potential can evolve concurrently with virus-induced chronic inflammation. Cancer Res; 77(13); 3591-604. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Vesicular stomatitis virus (VSV) is an oncolytic virus which selectively infects and kills cancer cells. The goal of the present study was to determine the safety and efficacy of VSV treatment of prostate tumors that arise in situ in immunocompetent, transgenic prostate-specific PTEN-null (PTEN(-/-)) mice. Interferon-sensitive VSV(AV3 strain), which expresses luciferase, was injected intraprostatically into tumor-bearing PTEN(-/-) and control mice and then monitored for tissue bioluminescence over 96 hours. Virus readily dispersed throughout the bodies of mice after only 3 hours; however, it persisted at high levels for >72 hours in PTEN(-/-) mice, but at relatively low levels and for only approximately 48 hours in controls. Plaque assays provided a similar pattern, with much higher concentrations of replicating virus in prostates of PTEN(-/-) mice than in controls. Transient, low levels of virus were detected in the spleens of both groups. Apoptotic analyses by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining revealed that VSV(AV3) is able to selectively infect and kill prostate cells in PTEN(-/-) mice, while sparing normal cells in control mice. The primary mechanism for cell kill is apparently apoptotic oncolysis as opposed to neutrophil invasion as has been reported using xenograft models. These results suggest that control of locally advanced human prostate cancer may be achievable through intraprostatic injection and amplification of a safe oncolytic virus, such as VSV(AV3).

EBV-associated gastric carcinoma is a distinct subset of gastric carcinoma infected with EBV, which shows latency I type expression of EBV latent genes (EBNA1, EBER, BARF0, and LMP2A). To clarify the role of EBV in this type of gastric carcinoma, the cell biological characteristics (growth, apoptosis, and migration) were evaluated in gastric carcinoma cell lines (MKN-1, TMK1, MKN-74 and MKN-7) with and without infection of recombinant EBV harboring the neomycin resistance gene. The infection reiterated the latency I type infection, and the only difference observed in EBV-infected gastric carcinoma cell lines was the resistance to serum deprivation-induced apoptosis. Comparative analyses of transcripts of apoptosis-associated genes in MKN-1 and EBV-MKN-1 and subsequent quantitative reverse transcription-PCR analysis showed up-regulation of the cellular survivin gene in EBV-infected gastric carcinoma cell lines. Small interfering RNA-mediated knockdown of survivin increased apoptosis in EBV-MKN-1 to the level of the original MKN-1 cells. Transfection of EBV-latent genes into MKN-1 showed that LMP2A, but not EBNA1, EBER, or BARF0, up-regulated survivin gene expression. LMP2A-mediated survivin up-regulation in gastric carcinoma cells was inhibited with a nuclear factor-kappaB (NF-kappaB) inhibitor, Bay 11-7082. In parallel with these findings in vitro, survivin expression was frequent in carcinoma tissues of gastric carcinoma by immunohistochemistry, and significantly more in EBV-associated gastric carcinoma (12 of 13) than in EBV-negative gastric carcinoma in the advanced stage (P = 0.0307). Thus, EBV uses its latent protein, LMP2A, to activate the NF-kappaB-survivin pathway to rescue EBV-infected epithelial cells from serum deprivation, and up-regulation of survivin may play a role in the progression of this specific type of gastric carcinoma infected with EBV.

Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer.

Nasopharyngeal carcinoma (NPC), an EBV-associated malignancy, is highly metastatic compared with other head and neck tumors, perhaps because of its viral link. Here, we show that the principal EBV oncoprotein, latent membrane protein 1 (LMP1), induces epithelial-mesenchymal transition (EMT) via Twist, a master transcriptional regulator in embryogenesis and newly implicated in metastasis, which, in turn, are likely to contribute to the highly metastatic character of NPC. LMP1 could induce EMT and its associated cell motility and invasiveness in a cell culture model, whereas expression of Twist small interfering RNA reversed LMP1-induced EMT. In diverse EBV-infected cell lines, expression of Twist correlates with expression of LMP1. Dominant-negative LMP1 could suppress Twist expression in EBV-positive cells, whereas LMP1 could induce Twist in EBV-negative nasopharyngeal cells. LMP1 signals through the nuclear factor-kappaB pathway, and an IkappaB superrepressor inhibited induction of Twist by LMP1. Finally, in human NPC tissues, expression of Twist and LMP1 is directly correlated and expression of Twist is associated with metastasis clinically. These results suggest that induction of Twist by a human viral oncoprotein LMP1 directly contributes to the metastatic nature of NPC.

Expression of the Coxsackie and Adenovirus Receptor (CAR) is frequently reduced in carcinomas, resulting in decreased susceptibility of such tumors to infection with therapeutic adenoviruses. Because CAR participates physiologically in the formation of tight-junction protein complexes, we examined whether molecular mechanisms known to down-regulate cell-cell adhesions cause loss of CAR expression. Transforming growth factor-beta (TGF-beta)-mediated epithelial-mesenchymal transition (EMT) is a phenomenon associated with tumor progression that is characterized by loss of epithelial-type cell-cell adhesion molecules (including E-cadherin and the tight junction protein ZO-1), gain of mesenchymal biochemical markers, such as fibronectin, and acquisition of a spindle cell phenotype. CAR expression is reduced in tumor cells that have undergone EMT in response to TGF-beta. This down-regulation results from repression of CAR gene transcription, whereas altered RNA stability and increased proteasomal protein degradation play no role. Loss of CAR expression in response to TGF-beta is accompanied by reduced susceptibility to adenovirus infection. Indeed, treatment of carcinoma cells with LY2109761, a specific pharmacologic inhibitor of TGF-beta receptor types I and II kinases, resulted in increased CAR RNA and protein levels as well as improved infectability with adenovirus. This was observed in cells induced to undergo EMT by addition of exogenous TGF-beta and in those that were transformed by endogenous autocrine/paracrine TGF-beta. These findings show down-regulation of CAR in the context of EMT and suggest that combination of therapeutic adenoviruses and TGF-beta receptor inhibitors could be an efficient anticancer strategy.

The adenoviral mutant dl922-947 has potent activity in a variety of tumors. We investigated the efficacy of dl922-947 in ovarian carcinoma; compared its activity to wild-type adenovirus, dl309, and dl1520; and investigated the use of icodextrin to enhance activity in vivo. We also assessed the utility of luciferase bioluminescence imaging to quantify the response of human ovarian carcinoma xenografts to dl922-947. Ovarian carcinoma cell lines were transfected in vitro with dl922-947, adenovirus 5 wild-type (Ad5 WT), dl309, and dl1520 and monitored for S-phase induction, viral protein expression, replication, and overall survival. In vivo, the efficacy of dl922-947 when delivered in PBS or icodextrin to female nude mice bearing IGROV1 xenografts was determined. In vitro, dl922-947 induced lysis with greater efficacy than Ad5 WT, dl309, or dl1520 in all ovarian carcinoma cell lines tested, which was associated with earlier expression of viral proteins and S-phase induction. The lytic effect in immortalized ovarian surface epithelial cells confirmed that cellular retinoblastoma pathway status is a strong determinant of dl922-947 activity. In vivo, i.p. delivery of dl922-947 (5 x 10(9) particles daily x 5) increased median survival from 20 to 96 days (P < 0.0001) and delivery in icodextrin-enhanced survival further. However, delayed hepatic toxicity was evident in some dl922-947-treated mice, which was not dependent upon viral replication within tumor cells or the liver. dl922-947 has potency in ovarian carcinoma and i.p. delivery in icodextrin may enhance this activity. Immunocompetent models of ovarian carcinoma are required for further evaluation of hepatotoxicity.

The idea of using oncolytic viruses for the treatment of cancers was proposed a century ago. During the last two decades, viruses able to replicate specifically in cancer cells and to induce their lysis were identified and were genetically modified to improve their viro-oncolytic properties. More recently, a new approach consisting of inducing selective apoptosis in cancer cells through viral infection has been proposed; this approach has been called viro-oncoapoptosis. In the present study, we report the property of bovine herpesvirus-4 (BoHV-4) to induce, in vitro and in vivo, apoptosis of some human carcinomas. This conclusion relies on the following observations: (a) In vitro, BoHV-4 infection induced apoptosis of A549 and OVCAR carcinoma cell lines in a time- and dose-dependent manner. (b) Apoptosis was induced by the expression of an immediate-early or an early BoHV-4 gene, but did not require viral replication. (c) Cell treatment with caspase inhibitors showed that apoptosis induced by BoHV-4 relied mainly on caspase-10 activation. (d) Infection of cocultures of A549 or OVCAR cells mixed with human 293 cells (in which BoHV-4 does not induce apoptosis) showed that BoHV-4 specifically eradicated A549 or OVCAR cancer cells from the cocultures. (e) Finally, in vivo experiments done with nude mice showed that BoHV-4 intratumoral injections reduced drastically the growth of preestablished A549 xenografts. Taken together, these results suggest that BoHV-4 may have potential as a viro-oncoapoptotic agent for the treatment of some human carcinomas. Moreover, further identification of BoHV-4 proapoptotic gene(s) and the cellular pathways targeted by this or these gene(s) could lead to the design of new cancer therapeutic strategies.

EBV is an etiological agent in infectious mononucleosis, is implicated in some malignant lymphomas and lymphoepithelioma-like carcinomas, and has been sporadically reported in carcinomas of the breast, lung, and other sites. We studied immunohistochemically benign and malignant tumors of the breast, lung, colon, and prostate and found EBV in some carcinomas of those sites. Also, EBV reactions were noted in hyperplasias and dysplasias, e.g., breast carcinomas in situ and prostatic intraepithelial neoplasia. Benign tumor counterparts were negative. PCR analysis of selected cases confirmed the presence of EBV. Our results suggest that EBV is not restricted to lymphoepithelioma-like carcinomas but may play an oncogenic role in frequent epithelial cancers and possibly also in hyperplasias and certain dysplasias preceding carcinomas.

EBV is an oncogenic herpesvirus associated with a number of human malignancies. The consistent presence of the EBV genome in certain tumors offers the potential for novel EBV-targeted therapies. EBV can infect cells in either a latent or lytic form. Here we demonstrate that a variety of chemotherapeutic agents, including cis-platinum, 5-fluorouracil (5-FU), and taxol, induce the switch from the latent to lytic form of EBV infection in tumor cells. This effect requires the protein kinase C delta, phosphatidylinositol 3'-kinase, and p38 stress mitogen-activated protein kinase signaling pathways but not caspase 3 activation. Because the lytic but not latent form of EBV infection converts the cytotoxic prodrug, ganciclovir (GCV), into its active form, we examined whether the combination of GCV and chemotherapy is more effective than chemotherapy alone for killing EBV-positive tumor cells. GCV significantly enhanced the ability of 5-FU and cis-platinum to kill EBV-positive, but not EBV-negative, gastric carcinoma cells in vitro. Most importantly, the combination of GCV and 5-FU (or GCV and cis-platinum) was much more effective in the treatment of EBV-positive nasopharyngeal carcinomas passaged in nude mice than either agent alone. These data suggest that GCV enhances the efficacy of conventional chemotherapy for the treatment of EBV-positive epithelial cell tumors.

EBV is found to be associated with 100% of poorly or undifferentiated nasopharyngeal carcinomas, a tumor of epithelial origin. The latent membrane protein-1 (LMP1) of EBV, may play a causal role in the development of this disease. The experiments initiated here were designed to examine the activity of LMP1 in vivo in the epidermis of PyLMP1 transgenic mice in relation to its putative role in carcinogenesis. Transgenic positive epidermis showed a 2-3-fold increase in the mitotic index, coupled with an increased level of expression of proliferative cytokeratin markers (K6 and K14) over controls. These results provide direct evidence that LMP1 induces proliferation in otherwise normal epithelial cells in vivo. To assess the role of LMP1 in tumorigenic progression, transgenic mice were treated topically with chemical carcinogens. PyLMP1 mice were highly sensitive to chemical carcinogens, developing significantly more small papillomas at a faster rate than controls. Furthermore, LMP1 could substitute for 12-O-tetradecanoylphorbol-13-acetate treatment in tumor promotion. However, LMP1 inhibited expansion of the benign lesions and did not enhance progression of the lesions to carcinomas or the progression of these to the more malignant spindle cell carcinomas. These data demonstrate that, early in the carcinogenic process, LMP1 acts as a tumor promoter after chemical initiation; but, paradoxically, it may also introduce a hurdle against expansion or progression of a lesion.

T-cell-mediated immune responses against oncogenic human papillomaviruses (HPVs) are believed to play a role in the prevention of cervical carcinogenesis. The in vitro production of interleukin 2 by CD4+ T helper (Th) cells in response to overlapping 20-mer peptides covering the HPV-16 E7 oncoprotein sequence was determined in 72 women with cytological evidence of premalignant cervical intraepithelial neoplasia (CIN) who participated in a nonintervention follow-up (FU) study. In addition, 15 HPV-16 + cervical carcinoma patients were tested. Positive Th cell reactivity was restricted to patients infected by HPV-16 and related types and showed a strong association with viral persistence and disease progression, as evidenced by the high frequency of positive responders among women with persistent HPV-16 infections who ended FU with high-grade CIN III lesions [14 of 15 (93%)]. Women with cervical carcinoma showed responses at a significantly reduced rate [7 of 15 (47%); P = 0.014]. Over the FU period (10-34 months), the level of E7-induced interleukin 2 production from the lymphocytes of CIN patients who had cleared HPV-16 infection showed an inverse correlation with time relative to the last positive HPV DNA test, with 8 of 13 of these patients showing positive responses after clearance. By contrast, among women with persistent HPV-16 infections and developing CIN III lesions (n = 8), there was a rise in Th cell activity over the course of FU. The majority of women responded to an immunogenic region in the carboxyl terminus of the E7 protein (amino acids 67-98). The observed HPV-16 E7-specific Th cell responses may develop as a consequence of increased antigen availability resulting either from clearance or from progression of cervical lesions.

The effect of the antiviral agent (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (cidofovir) on the EBV-associated tumor nasopharyngeal carcinoma (NPC) was evaluated in NPC xenografts in athymic mice. Intratumoral injection arrested tumor growth within 1 week, and by 4 weeks, tumors regressed to 8-75% (39 +/- 33%) of the original size, whereas control tumors injected with PBS grew to 282 +/- 25% of the original size. Ganciclovir slowed but did not arrest or cause regression of tumor growth. A striking antitumor effect was also produced by systemic administration; at 4 weeks, tumors were 79 +/- 49% of the original size, compared with 635 +/- 91% for the controls. Widespread apoptosis was detected after treatment for 2-6 days in C15 as well as two other NPC xenografts, C17 and C18; the latter NPCs have mutations in the p53 gene. These data indicate that cidofovir induces rapid cell death through apoptosis in EBV-transformed epithelial cells.

Specific human papilloma virus (HPV) types appear to be necessary etiological factors for most cervical cancers, yet additional genetic alterations seem to be required for their development and progression. The aim of this study is to determine the likely chromosomes location of tumorigenicity suppressor-like genes, the loss of function of which might be important in the origin or progression of cervical carcinomas. PCR with primers for 75 highly polymorphic microsatellite loci located on the major autosome arms were used to estimate the incidence of loss of heterozygosity (LOH) in 38 tumors. The HPV status of the tumors was also determined. LOH was found to involve 19 chromosome arms in 20-43% of the tumors. Chromosome arms 6p, 3p, and 18q are most frequently involved in LOH in 43, 39, and 35% of the informative carcinomas, respectively. The respective regions involved are 6p21.1-23, 3p13-25.3, and 18q12.2-21.2. LOH is generally limited to specific band segments within these regions. Similar high incidences of LOH of the same 3p segments have been reported in cervical carcinomas from different parts of the world. The same 3p and 6p segments are involved in many types of common cancers, whereas 18q changes are less frequent in other cancers. Chromosome arms 1q, 2q, 3q, 4p, 4q, 5p, 5q, 6q, 7q, 8p, 8q, 11q, 13q, 16p, 18p, and 19p are involved in LOH in 20-33% of the cervical tumors. Chromosome 11 alterations are among the most frequently found in many different types of neoplasias. In this study, 11p was involved in 16% of the tumors, and 11q was involved in 22%. Chromosome 17 alterations are found in more cancers than those of any other chromosome, frequently involving the p53 gene on 17p. LOH of 17p was found in 5 (15%) cervical tumors; 2 of these were HPV negative and expressed mutant p53. In such HPV-negative tumors, direct mutation of the wild-type p53 appears to replace the inactivation of the p53 product by oncogenic HPV types. Tumors with LOH at many loci were, on the average, at more advanced stages, as were tumors with mutant p53. The higher overall incidence of LOH in cervical carcinomas as compared to other cancers, and the diversity of LOH patterns found, suggest that different cervical carcinomas probably arise and/or progress, in part, because of the loss of function of different yet finite sets of tumorigenicity suppressor genes and genes that are involved in tumor progression and metastasis. The findings also indicate that certain chromosome segments that are often altered in cervical carcinomas are also frequently altered in several other types of cancers. It remains to be determined whether the same or different genes located within these segments are involved in the different cancer types.