Kenneth Garson

17β-Estradiol Accelerates Tumor Onset and Decreases Survival in a Transgenic Mouse Model of Ovarian Cancer

Epithelial ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE) but often goes undetected in the early stages, and as a result, the factors that contribute to its initiation and progression remain poorly understood. Epidemiological studies have suggested that the female steroid hormones are involved in ovarian carcinogenesis and that women who use hormone replacement therapy are at increased risk of developing the disease. A novel transgenic mouse model of ovarian cancer (tgCAG-LS-TAg) was developed to examine the role of the female reproductive steroid hormones [17beta-estradiol (E(2)) and progesterone (P(4))] on the initiation, progression, and pathology of ovarian cancer. The mouse model uses the Cre-LoxP system to induce expression of the simian virus 40 large and small T antigens (SV40 TAg). After targeted induction of the oncogene in the OSE, mice develop poorly differentiated ovarian tumors, tumor dissemination to tissues within the abdominal cavity, and a subset develops hemorrhagic ascites. Treatment with P(4) had no impact on the disease, but E(2) altered the pathophysiology, resulting in an earlier onset of tumors, decreased overall survival time, and a distinctive papillary histology. Normal ovaries collected from mice treated with E(2), but lacking expression of SV40 TAg, displayed an increase in the areas of columnar and hyperplastic OSE cells compared to placebo-treated controls. A better understanding of the mechanisms by which E(2) alters the morphology of normal OSE cells and reduces survival in this mouse model may translate into improved prevention and treatment options for women using hormone replacement therapy.

Models of ovarian cancer—Are we there yet?

Ovarian cancer is the most lethal of all gynecological cancers and arises most commonly from the surface epithelium. Successful clinical management of patients with epithelial ovarian cancer is limited by the lack of a reliable and specific method for early detection, and the frequent recurrence of chemoresistant disease. Experimental models are of crucial importance not only to understand the biological and genetic factors that influence the phenotypic characteristics of the disease but also to utilize as a basis for developing rational intervention strategies. Ovarian cancer cell lines derived from ascites or primary ovarian tumors have been used extensively and can be very effective for studying the processes controlling growth regulation and chemosensitivity or evaluating novel therapeutics, both in vitro and in xenograft models. While our limited knowledge of the initiating events of ovarian cancer has restricted the development of models in which the early pathogenic events can be studied, recent advances in the ability to manipulate gene expression in ovarian surface epithelial cells in vitro and in vivo have begun to provide insights into the molecular changes that may contribute to the development of ovarian cancer. This review highlights the strengths and weaknesses of some of the current models of ovarian cancer, with special consideration of the recent progress in modeling ovarian cancer using genetically engineered mice.

Harnessing Oncolytic Virus-mediated Antitumor Immunity in an Infected Cell Vaccine

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte-monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSVs extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.

Epithelial ovarian cancer stem cells: underlying complexity of a simple paradigm

The lack of significant progress in the treatment of epithelial ovarian cancer (EOC) underscores the need to gain a better understanding of the processes that lead to chemoresistance and recurrence. The cancer stem cell (CSC) hypothesis offers an attractive explanation of how a subpopulation of cells within a patients tumour might remain refractory to treatment and subsequently form the basis of recurrent chemoresistant disease. This review examines the literature defining somatic stem cells of the ovary and fallopian tube, two tissues that give rise to EOC. In addition, considerable research has been reviewed, that has identified subpopulations of EOC cells, based on marker expression (CD133, CD44, CD117, CD24, epithelial cell adhesion molecule, LY6A, ALDH1 and side population (SP)), which are enriched for tumour initiating cells (TICs). While many studies identified either CD133 or CD44 as markers useful for enriching for TICs, there is little consensus. This suggests that EOC cells may have a phenotypic plasticity that may preclude the identification of universal markers defining a CSC. The assay that forms the basis of quantifying TICs is the xenograft assay. Considerable controversy surrounds the xenograft assay and it is essential that some of the potential limitations be examined in this review. Highlighting such limitations or weaknesses is required to properly evaluate data and broaden our interpretation of potential mechanisms that might be contributing to the pathogenesis of ovarian cancer.

A New Spontaneously Transformed Syngeneic Model of High-Grade Serous Ovarian Cancer with a Tumor-Initiating Cell Population

Improving screening and treatment options for patients with epithelial ovarian cancer has been a major challenge in cancer research. Development of novel diagnostic and therapeutic approaches, particularly for the most common subtype, high-grade serous ovarian cancer (HGSC), has been hampered by controversies over the origin of the disease and a lack of spontaneous HGSC models to resolve this controversy. Over long-term culture in our laboratory, an ovarian surface epithelial (OSE) cell line spontaneously transformed OSE (STOSE). The objective of this study was to determine if the STOSE cell line is a good model of HGSC. STOSE cells grow faster than early passage parental M0505 cells with a doubling time of 13 and 48 h, respectively. STOSE cells form colonies in soft agar, an activity for which M0505 cells have negligible capacity. Microarray analysis identified 1755 down-regulated genes and 1203 up-regulated genes in STOSE compared to M0505 cells, many associated with aberrant Wnt/β-catenin and Nf-κB signaling. Upregulation of Ccnd1 and loss of Cdkn2a in STOSE tumors is consistent with changes identified in human ovarian cancers by The Cancer Genome Atlas. Intraperitoneal injection of STOSE cells into severe combined immunodeficient and syngeneic FVB/N mice produced cytokeratin+, WT1+, inhibin−, and PAX8+ tumors, a histotype resembling human HGSC. Based on evidence that a SCA1+ stem cell-like population exists in M0505 cells, we examined a subpopulation of SCA1+ cells that is present in STOSE cells. Compared to SCA1− cells, SCA1+ STOSE cells have increased colony-forming capacity and form palpable tumors 8 days faster after intrabursal injection into FVB/N mice. This study has identified the STOSE cells as the first spontaneous murine model of HGSC and provides evidence for the OSE as a possible origin of HGSC. Furthermore, this model provides a novel opportunity to study how normal stem-like OSE cells may transform into tumor-initiating cells.

Technical challenges and limitations of current mouse models of ovarian cancer.

The development of genetically engineered models (GEM) of epithelial ovarian cancer (EOC) has been very successful, with well validated models representing high grade and low grade serous adenocarcinomas and endometrioid carcinoma (EC). Most of these models were developed using technologies intended to target the ovarian surface epithelium (OSE), the cell type long believed to be the origin of EOC. More recent evidence has highlighted what is likely a more prevalent role of the secretory cell of the fallopian tube in the ontogeny of EOC, however none of the GEM of EOC have demonstrated successful targeting of this important cell type.The precise technologies exploited to develop the existing GEM of EOC are varied and carry with them advantages and disadvantages. The use of tissue specific promoters to model disease has been very successful, but the lack of any truly specific OSE or oviductal secretory cell promoters makes the outcomes of these models quite unpredictable. Effecting genetic change by the administration of adenoviral vectors expressing Cre recombinase may alleviate the perceived need for tissue specific promoters, however the efficiencies of infection of different cell types is subject to numerous biological parameters that may lead to preferential targeting of certain cell populations.One important future avenue of GEM of EOC is the evaluation of the role of genetic modifiers. We have found that genetic background can lead to contrasting phenotypes in one model of ovarian cancer, and data from other laboratories have also hinted that the exact genetic background of the model may influence the resulting phenotype. The different genetic backgrounds may modify the biology of the tumors in a manner that will be relevant to human disease, but they may also be modifying parameters which impact the response of the host to the technologies employed to develop the model.

A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses

Background Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value. Methodology/Principal Findings Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene. Conclusion/Significance The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.

Microtubule disruption synergizes with oncolytic virotherapy by inhibiting interferon translation and potentiating bystander killing

In this study, we show that several microtubule-destabilizing agents used for decades for treatment of cancer and other diseases also sensitize cancer cells to oncolytic rhabdoviruses and improve therapeutic outcomes in resistant murine cancer models. Drug-induced microtubule destabilization leads to superior viral spread in cancer cells by disrupting type I IFN mRNA translation, leading to decreased IFN protein expression and secretion. Furthermore, microtubule-destabilizing agents specifically promote cancer cell death following stimulation by a subset of infection-induced cytokines, thereby increasing viral bystander effects. This study reveals a previously unappreciated role for microtubule structures in the regulation of the innate cellular antiviral response and demonstrates that unexpected combinations of approved chemotherapeutics and biological agents can lead to improved therapeutic outcomes.

Generation of Tumors in Transgenic Mice Expressing the SV40 T Antigen Under the Control of Ovarian-Specific Promoter 1:

Objective: The ovarian-specific promoter, OSP-1, which was cloned from the transcript of a rat retrovirus-like element specifically expressed in ovarian tissue, was tested for its ability to drive ovary-specific transcription in transgenic mice. Methods: Transgenic mice were generated with the lacZ reporter gene (OSP-lacZ) or the early region of SV40 virus (OSP-TAg) placed under the control of the OSP-1 promoter. OSP-lacZ and OSP-TAg transgenic animals were examined, respectively, for the expression of lacZ (OSP-lacZ) or the development of tumors (OSP-TAg). Results: The expression of lacZ in the resulting OSP-lacZ mice was restricted to the ovary as determined by X-gal staining of multiple organs. Immunohistochemical detection of β-galactosidase showed lacZ expression mainly in the granulosa cells and ovarian surface epithelial cells. OSP-TAg mice developed tumors in a variety of tissues, including unilateral granulosa cell tumors in two of three female founder mice. In the contralateral ovary of one mouse with a granulosa cell tumor, there wee alterations in the ovarian surface epithelial cells suggestive of preneoplasia. Conclusions: Although the OSP-1 promoter was able to restrict reporter gene expression to the ovary in transgenic mice, the expression of TAg in the OSP-TAg mice resulted in ovarian tumors as well as tumors in numerous other organs. This indicated that although transcription from the OSP-1 promoter occurs predominantly in the ovary, this promoter is sufficiently leaky in cells in other tissues to permit their tumorigenic conversion by SV40 TAg.

The N-terminal env-derived amino acids of v-rel are required for full transforming activity

Expression of the v-rel oncogene of the reticuloendotheliosis virus, strain T (REV-T), can mediate the transformation of chicken spleen and bone marrow cells. Although the majority of the coding sequence of the v-rel oncogene is derived from the cellular rel sequence, the N- and C-terminal amino acids are coded for by remnants of the REV env gene. The resulting v-rel protein can be described as an env-rel-(out of frame env) fusion protein. Terminal deletion mutants were constructed to determine the role that env sequences play in the transforming activity of v-rel. Deletions were designed to remove only sequences of v-rel derived from former env sequence. Additional deletions removed more substantial amounts of coding sequence. Introduction of deleted genes into an REV-T based retroviral vector permitted the transforming activities to be determined. Deletion analysis indicated that the N-terminal region of pp59v-rel is required for the transforming activity, whereas as many as 100 C-terminal amino acids could be deleted without complete loss of the activity.