Models of ovarian cancer—Are we there yet?

Molecular and Cellular Endocrinology 239 (2005) 15–26

At the Cutting Edge

Models of ovarian cancer—Are we there yet? Kenneth Garson a,∗ , Tanya J. Shaw a,b , Katherine V. Clark a,b , De-Sheng Yao c , Barbara C. Vanderhyden a,b,d a Centre for Cancer Therapeutics, Ottawa Health Research Institute, 503 Smyth Road, Ottawa, Ont., Canada K1H 1C4 Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ont., Canada K1H 8M5 Guangxi Cancer Institute, Affiliated Tumor Hospital, Guangxi Medical University, 6 BinHu Road, Nanning, Guangxi 530021, PR China d Department of Obstetrics and Gynecology, University of Ottawa, 501 Smyth Road, Ottawa, Ont., Canada K1H 8L6 b

c

Received 10 March 2005; received in revised form 29 March 2005; accepted 30 March 2005

Abstract 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. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovarian cancer; Xenograft models; Cell transformation

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exploring the diversity of human ovarian cancers in xenograft models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modeling early ovarian cancer with ovarian surface epithelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Human OSE cell transformation: choice of oncogene delineates phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Mouse OSE cell transformation: does the immune system make a difference? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic mouse models: is the MISIIR promoter the answer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exploiting the Cre–loxP recombination system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Are we there yet? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗

Corresponding author. E-mail addresses: [email protected] (K. Garson), [email protected] (T.J. Shaw), [email protected] (K.V. Clark), [email protected] (D.-S. Yao), [email protected] (B.C. Vanderhyden). 0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.03.019

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1. Introduction Epithelial ovarian cancer has less than a 1% life-time risk, yet is the most lethal of the gynecologic malignancies (American Cancer Society, 2003). This is due in part to the late diagnosis following a commonly asymptomatic early disease, as well as the high rate of chemo-resistance, which limits treatment of recurrent disease. Current research efforts are focused on improving our understanding of the etiology and early stages of the disease, as well as in the development and evaluation of novel therapeutics. While the ultimate goal of ovarian cancer models is to provide a system for the discovery and testing of novel therapeutics, the process of developing and refining models to more accurately reflect human ovarian cancer forces us to understand the disease in greater detail. The past 5 years have yielded remarkable progress in the development of models of ovarian cancer, including in vitro transformed human and murine ovarian surface epithelial cells, xenotransplanted cancer cells with defined histopathological features, and the first transgenic model of epithelial ovarian cancer. From these models has come the observation that inactivation/activation of different signalling pathways can lead to the emergence of ovarian cancers with different phenotypes and histologies. While each of these models has unique strengths, there remains significant restrictions on their relevance to human disease. Tumor development from xenografted cells is achieved only in immune-compromised animals. The degree to which in vitro transformation of human and mouse cells faithfully represents the process in vivo remains unknown. Early onset of tumor development in the transgenic model precludes investigation of mechanisms of disease initiation or preventive interventions. In addition, the inability to simply and accurately monitor tumor progression of intraperitoneal disease remains a challenge for all models. Despite these limitations, the recent advances in model development have brought much excitement to the study of ovarian cancer, and the application of genetics and targeting approaches will undoubtedly improve and refine these models and their relevance to human ovarian cancer. This review examines xenograft, cell culture and transgenic models of ovarian cancer with particular emphasis on recent advances and remaining challenges.

2. Exploring the diversity of human ovarian cancers in xenograft models Xenografting human cancer cells into immune-deficient mice has been a popular and useful research tool since the late 1960s (Rygaard and Povlsen, 1969), and has been essential for analyzing the tumorigenicity of cells, the histology of tumors that they form, and in the evaluation of therapeutics. Human ovarian cancer cell xenografts are possibly the most widely used research models for the study of ovarian cancer as they are derived from naturally occurring clinical cases, and are therefore most representative of the diverse disease.

Ovarian cancer xenografts can entail inoculation of the tumor cells subcutaneously (SC), intraperitoneally (IP) or orthotopically. Rodents have a unique bursal membrane that surrounds the ovary that facilitates orthotopic injection of cells, which is important as the ovarian microenvironment has been identified to influence cancer cell behavior (Shaw et al., 2004). Though useful in confining cancer cells to the ovary and providing a strategy to model early stage disease, the bursal membrane may limit the value of these models in the study of the later stages of ovarian cancer progression. The bursa, absent in humans, may act as a physical barrier to cancer cell dissemination and metastasis. Intraperitoneal injection of cancer cells, however, may accurately model advanced disease, as ovarian cancer metastases frequently appear disseminated throughout the peritoneum. A disadvantage of the intrabursal and IP models is the difficulty in monitoring disease progression. It is possible to visualize tumor burden with magnetic resonance imaging (Gossmann et al., 2000) or with cells that stably express green fluorescent protein (Chaudhuri et al., 2001); however, the need to transport immunodeficient animals repeatedly from their protective environments to the equipment for regular monitoring of tumor growth can be problematic. Some studies have successfully monitored serum markers such as secreted alkaline phosphatase (Nilsson et al., 2002), and CA125 (Burbridge et al., 1999; Kraeber-Bodere et al., 1996), which can be easily evaluated by ELISA and may also be a useful tool for assessing tumor burden. A second important drawback of xenograft models of ovarian cancer is that they rely on the immunodeficiency of the recipient animals, which has the potential to affect tumor formation, as well as therapeutic response. There are five main histological subtypes of ovarian cancer (serous, mucinous, clear cell, endometrioid and transitional cell/Brenner tumors) as well as undifferentiated adenocarcinomas (Scully, 1995), and it is thought that each subtype differs in its etiology and genetic aberrations (Chatzistamou et al., 2002; Tsukagoshi et al., 2003). Attempts to personalize treatment regimens with relevant targeted therapeutics must be preceded by pre-clinical drug evaluations in cancer cell lines for which characteristics such as histology and gene expression profiles are well understood. Some exemplary original references, as well as our recent study characterizing the histopathology of tumors resulting from commonly used ovarian cancer cell lines, provide details of the tumor subtype that should be considered in the interpretation and extrapolation of results from these unique models. For instance, HEY cell lines were derived from a moderately differentiated papillary cystadenocarcinoma (Buick et al., 1985), but form undifferentiated carcinomas when xenografted into nude mice (Shaw et al., 2004); OV-MZ 1-6 are recognized as serous cystadenocarcinomas (Mobus et al., 1992); both the A2780 and H134 cells have been reported to form undifferentiated tumors; OVCAR3 form poorly differentiated serous adenocarcinomas (Molthoff et al., 1991); and SKOV3 tumors were clear cell carcinomas (Shaw et al., 2004). We have shown that despite similar morphologies of OVCA 429 and OV2008

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Fig. 1. Human ovarian cancer cell lines and corresponding intraperitoneal xenograft tumors. OVCA 429 cells in culture (A) and following intraperitoneal injection, which resulted in the formation of papillary adenocarcinoma (B). OV2008 cells in culture (C) and in vivo after intraperitoneal inoculation of tumor cells. Tumors of endometrioid differentiation were produced (D). Magnification: ×200.

cells in vitro, similar rates of tumor progression as xenografts (median survival of 62 days versus 66 days, respectively), and similar presentation of large, solid tumors, OVCA 429 produced papillary adenocarcinomas with clear cell characteristics, whereas OV2008 formed tumors of endometrioid differentiation (Fig. 1 and Shaw et al., 2004). It is clear that the choice of cell line used in xenograft models should be made with the appreciation that different ovarian cancer cell lines may model different histological tumor subtypes, which in turn may demand different therapeutic approaches. The use of human ovarian cancer cell lines must include an awareness that the cell lines used for these models represent tissue culture outgrowths of late stage ovarian cancer. The genetic changes resident in these cell lines may represent a blend of genetic alterations selected in the patient during progression and treatment of their disease, combined with further genetic changes selected during their adaptation to tissue culture. Also, with the demonstrated role of tumor stem cells in other cancer models (Behbod and Rosen, 2005), it is important to determine whether human ovarian tumors are also fuelled by cancer stem cells. If tumor stem cells play a role in ovarian cancer, a significant improvement in cell culture and xenograft models may result from the isolation and culture of such cells.

Studies combining xenograft models with the use of RNA interference to down-regulate gene expression will allow a detailed dissection of the roles that various signalling pathways play in the malignant phenotype of these cell lines (Yang et al., 2003). Such analysis has the potential to strengthen our knowledge of altered signalling required for tumorigenesis, and also to identify novel therapeutic targets.

3. Modeling early ovarian cancer with ovarian surface epithelial cells While xenograft models using human cancer cell lines may shed light on the nature and behavior of advanced human ovarian cancers, there is great interest in understanding the earlier steps of ovarian cancer and to develop models of ovarian cancer which will reflect both early and late stages of disease. The ability to isolate and culture relatively pure populations of ovarian surface epithelial (OSE) cells from human (Auersperg et al., 1984; Gregoire et al., 1998; Nitta et al., 2001; Tsao et al., 1995) and non-human sources (Adams and Auersperg, 1981; Godwin et al., 1992; Hoffman et al., 1993; Kido and Shibuya, 1998; Roby et al., 2000) has given investigators the opportunity to systematically genetically modify these normal cells in order to define their requirements for

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tumorigenic transformation. Most importantly, use of these in vitro model systems has begun to delineate the contributions of specific oncogenes to tumor subtype. Important questions arise with respect to the validity of tumor models based on in vitro cell cultures. Are cultures of ovarian surface epithelial cells representative of the normal epithelium resident on the ovarian surface? Does the malignant transformation of OSE in culture bear any relevance to the malignant transformation that occurs in clinical disease? Are there species differences that make human OSE more relevant as models than their mouse counterparts? In the case of xenografts or human OSE studies, does the ability to form tumors in immunodeficient animals fairly reflect the process in immune-competent animals? In the following text, we describe various OSE culture-based models of ovarian cancer and emphasize how these models address the above questions. Several early reports have shown that following repeated passages, normal murine OSE (MOSE) and rat OSE (ROSE) are able to transform spontaneously to a tumorigenic phenotype (Godwin et al., 1992; Roby et al., 2000; Testa et al., 1994). While these reports are not new, it is relevant to question whether the emergence of tumorigenic cells passaged under tissue culture conditions can shed any light on the processes leading to the development of human ovarian cancer. Analysis of the changes in gene expression between normal rat OSE and their malignant derivatives culminated in the identification of the lot1 tumor suppressor (Abdollahi et al., 1997) whose expression was found to be routinely downregulated in transformed ROSE. The discovery of lot1 in the rat model led to a more recent study showing lot1 expression to be severely reduced in 80% and undetectable in approximately 40% of human ovarian cancers (Cvetkovic et al., 2004). Therefore, the study of spontaneously transformed ROSE has allowed the identification of a tumor suppressor, which appears to play a role in human ovarian cancer. Although only a single example, the results do suggest that spontaneous transformation of OSE in culture has some relevance to the malignant transformation that occurs in clinical disease. More recent experiments defining the genetic events that are required for conversion of normal OSE to a tumorigenic phenotype have exploited both mouse and human cells, each species presenting certain advantages and disadvantages. Since murine and human OSE exhibit structural and physiological differences (Auersperg et al., 2001), studies with HOSE are useful as they avoid species differences from the disease that we are attempting to model. In addition, human cells in general appear to have greater barriers to malignant transformation than murine cells (Hahn and Weinberg, 2002). While inactivation of p53 is sufficient to bypass senescence in mouse embryo fibroblasts, human fibroblasts require inactivation of p53, the retinoblastoma gene (Rb), and the activation of telomerase to bypass senescence. Similarly, growth arrest induced by oncogenes such as Ha-ras can be abrogated by inactivation of p53 in murine

primary cells, whereas inactivation of both p53 and Rb is required in human cells. Finally, the key effectors of the Ras pathway important for transformation may differ between mouse and human (Hamad et al., 2002). Despite these differences, the models described below consistently affirm the important roles of p53, Rb and Ras in both mouse and human OSE cell transformation.

3.1. Human OSE cell transformation: choice of oncogene delineates phenotype In order to use HOSE for ovarian cancer models, investigators have tried various oncogenes to induce the immortalization and transformation process. Early passage HOSE are immortalized by transfecting in the early region of SV40 (LT) and the catalytic subunit of telomerase (hTERT) (Davies et al., 2003; Kusakari et al., 2003; Liu et al., 2004). As a point of clarification, for the purposes of this review, expression of the “large T antigen” also implies the expression of the alternatively spliced products small t antigen and the 17k t antigen. The resulting HOSE/LT/hTERT cells are immortal, with slightly enhanced anchorage-independent growth compared to normal HOSE, but are not tumorigenic in nude mice (Kusakari et al., 2003; Liu et al., 2004). The subsequent expression of activated Ha-ras or c-erbB2 in these cells failed to significantly increase their growth rate, but increased their anchorage-independent growth, increased their resistance to apoptosis induced by serum deprivation, and rendered them tumorigenic in nude mice (Kusakari et al., 2003). Subcutaneous injection of nude mice with OSE/LT/hTERT/Ha-ras cells or OSE/LT/hTERT/c-erbB2 cells resulted in the development of carcinomas in 40 and 50% of mice, respectively. Carcinomas induced by Ha-ras were positive for cytokeratin (CK) whereas tumors induced by c-erbB2, although of similar morphology, were positive for both CK and vimentin. It is to be noted that the acquisition of the tumorigenic phenotype was accompanied by significant increases in the levels of VEGF secreted in cell culture. Liu et al. (2004) described the tumorigenic transformation of the human OSE cell lines IOSE-80 and IOSE-29 following immortalization with hTERT and transfection with either activated Ha-ras or K-ras genes. The IOSE-80 and IOSE-29 cell lines (Auersperg et al., 1984, 1999) are normal HOSE whose lifespan has been extended by transfection with the early region of SV40. Both ras alleles transformed the IOSE80/LT/hTERT lines into poorly differentiated carcinomas following IP injection. The epithelial nature of the resulting tumors was confirmed by the detection of CK19 expression. Interestingly, the IOSE-29/LT/hTERT cell line transfected with Ha-ras formed sarcomas that, like the parental IOSE-29 cells, were negative for CK19 expression. In contrast, expression of K-ras in the IOSE-29/LT/hTERT cell line produced an IP tumor that showed a biphasic phenotype with regions of poorly differentiated sarcoma as well as CK19-positive carcinoma.

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Using microarray analysis of the changes in gene expression induced by the ras genes, Liu et al. (2004) went on to investigate the roles of IL-1␤ and IL-8, which were upregulated in their RAS-transfected cells. In serum-starved cultures, antibody neutralization of the secreted IL-1␤ and IL-8 increased the number of apoptotic cells in the RAStransfected cultures suggesting that in this model system, IL-1␤ and IL-8 may play a positive role in cell survival. In order to determine if RAS expression also coordinates the up-regulation of similar cytokines in naturally occurring human ovarian cancers, the over-expression of wild-type Haras was knocked down in the SKOV3 ovarian cancer cell line using RNA interference. SKOV3 cells with decreased Haras expression showed reduced expression of both IL-1␤ and IL-8 supporting the observations made from their genetically defined model system (Liu et al., 2004). These two studies by Kusakari et al. and Liu et al. demonstrate not only the ability of the authors to malignantly transform HOSE with potent oncogenes, they also point to more subtle outcomes. In both studies, the choice of oncogene led to differences in the phenotype of the emerging tumors. The c-erbB2 oncogene led to tumors which coexpressed vimentin with the epithelial cytokeratin markers, and K-ras but not Ha-ras was able to convert the IOSE29/LT/hTERT cell line into tumors with an epithelial component. This hints at the future of genetically modified OSE models; delineating the roles and subtle differences that various oncogenes play in the tumorigenic transformation of OSE. Also, both studies identified the up-regulation of factors, namely VEGF and interleukins, which might play an important role in the malignancy of the resulting tumors. In vitro culture of human OSE provides a convenient model for exploring early steps in epithelial ovarian cancer formation, and represents the only current model with which to study the events associated with transformation of human cells. However, there remain significant concerns with these models that must be considered, notably whether cultured cell lines accurately represent normal ovarian surface epithelium. Examination by cDNA microarray of gene expression in human ovarian cancer cell lines, immortalized HOSE, primary cultures of HOSE and HOSE harvested directly from scrapings from ovaries has indicated that even short-term culturing in vitro impacts dramatically on their gene expression profile (Zorn et al., 2003). In addition, since the early region of SV40 has transforming activities above and beyond the binding of the large T antigen to pRb and p53 (Sullivan and Pipas, 2002), it would be of benefit to determine if the down-regulation of pRb and p53 by short-hairpin RNA strategies in combination with the expression of hTERT is sufficient to render HOSE immortal. Finally, while this system has the advantage of working with “normal” human precursors, ultimately their tumorigencity can only be tested in immunodeficient mice, ignoring the role of the immune system on the possible outcomes of tumor progression and treatments.

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3.2. Mouse OSE cell transformation: does the immune system make a difference? Modification of oncogene and tumor suppressor function in MOSE allows for the possibility of studying disease progression and therapeutic response in immune-competent animals, an important aspect that is missing in mouse models receiving human cancer xenografts. In addition, an understanding of the roles of various oncogenes/tumor suppressors in converting MOSE to a tumorigenic phenotype would be of value for the development of future transgenic models. In our laboratory, we have employed a retroviral transduction protocol to introduce various oncogenes into MOSE cultures to screen for relevant combinations that might be useful in the generation of transgenic models. In examination of the c-myc, mutant k-ras and NeuNT (an activated mutant rat cerbB2) genes, we have found that a combination of k-ras plus c-myc results in the more rapid proliferation of MOSE and the most tumorigenic cells in nude mouse models (manuscript in preparation). While this system is convenient for the identification of putative oncogenes to target to the ovarian surface epithelium in transgenic mice, it does rely on primary cultures of MOSE that have adapted to growth in cell culture. Orsulic et al. (2002) used retroviral transduction to express combinations of oncogenes in MOSE with minimal time in culture. Explants of ovaries were cultured transiently in vitro to permit repeated retroviral infection over the course of 1 week prior to either subcutaneous injection or the reinsertion of the cells into the bursal space of ovariectomized nude mice. Elegantly, in two different experimental models, they were able to direct infection, and hence, oncogene expression either to all cell types in the ovary or exclusively to the surface epithelial cells. In their study using c-myc, kras or AKT, they concluded that the expression of at least two oncogenes and a p53-null background were required for efficient tumorigenesis in nude mice within an 8-week endpoint. Similar results were obtained when cells were injected orthotopically under the ovarian bursa, however tumors in this model metastasized throughout the peritoneum and produced bloody ascites. While the expression of single oncogenes was sufficient to cause tumors, their long latency (>12 weeks) was thought to reflect the need for secondary, undefined genetic changes. The emerging tumors in their experimental animals consistently expressed CK, confirming an epithelial phenotype, regardless of whether the viral vectors were infecting only the surface epithelium or all of the ovarian cell types. This suggested that the surface epithelial cells were most sensitive to transformation by the oncogenes tested, consistent with the clinical observation that most ovarian tumors are epithelial in nature and thought to be derived from the surface epithelium. Interestingly, when tested in immunecompetent syngeneic mice, cells transformed by k-ras, c-myc and AKT in a p53-null background produced tumors that were restricted to the site of injection under the bursal membrane with a much longer latency of three months. This supports suggestions that an immune-competent model of

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ovarian cancer may present additional hurdles to the initiation, progression and metastasis of experimental tumors. The use of murine OSE introduces the complication of species differences and, as in human, the concern that even short-term culturing may reprogram their gene expression. Despite these concerns, experimental models with murine OSE can include examination of tumorigenicity in immunecompetent animals and ultimately these experiments can lead to transgenic models in which tumors initiate and progress in situ.

4. Transgenic mouse models: is the MISIIR promoter the answer? One of the biggest challenges of creating a mouse model of ovarian cancer that adequately recapitulates the human disease has been not only identifying the appropriate genetic pathways to target, but determining how to manipulate these pathways in a manner that is spatially and temporally defined. The development of traditional transgenic models of ovarian cancer has been difficult due to the lack of suitable promoters that can drive transgene expression exclusively to the ovarian surface epithelium. Only two candidate promoters have been reported in association with attempts to generate models of epithelial ovarian cancer, and only one of these has been successful. Recently, we explored the utility of the ovarian-specific promoter 1 (OSP-1) (Selvakumaran et al., 2001) for the generation of a transgenic model of ovarian cancer (Garson et al., 2003). The OSP-1 promoter was able to specifically target the expression of ␤-galactosidase to the mouse ovary with expression noted mainly in the granulosa and surface epithelial cells. Unfortunately, expression in transgenic mice of the early region of SV40 (TAg) under the control of the OSP-1 promoter revealed a more “leaky” expression, which resulted in tumors appearing in multiple tissues in transgenic mice. Although ovarian tumors did arise, they appeared to have originated from the granulosa cell compartment. The first successful transgenic model of epithelial ovarian cancer was reported by Connolly et al. (2003). They noted earlier reports that the Mullerian inhibiting substance receptor type II (MISIIR) was up-regulated in ovarian cancer cell lines and patient ascites (Masiakos et al., 1999). After demonstrating that the mouse ovarian surface epithelium also expresses the MISIIR transcript, they generated transgenic mice expressing the early region of SV40 under the control of the MISIIR promoter (tgMISIIRTAg). Approximately 50% of the transgenic females developed bilateral ovarian tumors within 6–13 weeks of age. The tumors were frequently associated with peritoneal dissemination including the appearance of ascites. Although the endogenous MISIIR promoter shows strong expression in granulosa cells, the emerging tumors in the transgenic mice were poorly differentiated carcinomas, which stained positively for the expression of both CK8 and CK19.

We have found that tumors are already evident in the ovaries of newborn tgMISIIRTAg mice (Fig. 2A and B). This makes the tgMISIIRTAg model unsuitable for the analysis of the early lesions in the development of ovarian cancer and for the modulation of factors that might influence the initiation or onset of the disease. The epithelial nature of the tumors in the tgMISIIRTAg mice does make it a worthy model for further investigation of factors that might influence the progression of existing disease or for the testing of therapeutics. The development of future transgenic models of epithelial ovarian cancer will need to focus on delaying the onset or initiation of the disease, using inducible systems for example, to better reflect the clinical situation in patients. Once this is achieved, future models can then be tailored to exploit the activation of oncogenes and/or inactivation of tumor suppressors that are most relevant to human ovarian cancers.

5. Exploiting the Cre–loxP recombination system The difficulty in finding suitable promoters for conventional transgenic models of epithelial ovarian cancer has been circumvented by the exploitation of the Cre–loxP recombination system. Model systems based on this technology have significant advantages over the other model systems, and although only two have been reported thus far, these models have proven to be highly informative. The Cre–loxP system consists of cre, a recombinase, and its 34 bp binding site, loxP. Portions of DNA flanked on each side by loxP sites (floxed) are irreversibly excised from the genome following the action of cre recombinase. In practice, transgenic mice with floxed sequences can be designed to either remove important exons of the targeted gene to produce a conditional knock-out, or to remove transcriptional stop sequences (“lox-stop”) situated between a promoter and a transgene resulting in transcription and expression of the transgene upon expression of cre. Thus, these genes may only be switched on or off when the mouse is exposed to the cre recombinase, either via crossing with a mouse generated to express this enzyme, or via targeted delivery to somatic cells via a viral vector (Sauer, 1998). This system has been successfully employed in the development of mouse models of several different types of cancers (Meuwissen et al., 2001), in particular the modeling of initiation and progression of tumors in the breast epithelium (Xu et al., 1999). The Cre–loxP system has recently been used in the development of two different mouse models of ovarian cancer, which each targeted different genetic pathways. In both of these studies, adenoviral vectors expressing cre recombinase (AdCre) were injected under the ovarian bursal membrane, which encapsulates the ovary. Injection of AdCre under the bursal membrane permits the controlled activation of “loxstop” oncogenes or the inactivation of floxed tumor suppressors specifically in the surface epithelium. In the first of these studies, researchers chose to target the pathways of two major tumor suppressor genes, p53 and

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Fig. 2. Mouse ovarian tumors resulting from T antigen expression targeted to the ovary. (A) H&E section of an enlarged ovary from a 5 day tgMISIIRTAg mouse. Although oocytes are still evident, poorly differentiated tumor cells constitute the bulk of the ovary. The inset (bottom left) shows the normal ovary of a non-transgenic littermate control (same magnification, black bar represents 100 ␮m). (B) Immunohistochemical detection of TAg expression in the ovary of a 5-day-old tgMISIIRTAg mouse. Tissue along the left edge is the ovarian bursa. The black magnification bar represents 50 ␮m. (C) Expression of lacZ in tgCAG-LS-TAg mice. Mice carrying the tgCAG-LS-TAg transgene (top) express lacZ in a variety of organs including localized expression on the surface of the ovary (bottom right). Ovaries from tgCAG-LS-TAg mice (transgenic) and non-transgenic littermate controls were fixed, then stained with X-gal. Approximately 30% of tgCAG-LS-TAg mice develop epithelial ovarian tumors within 11–25 weeks following intrabursal AdCre injection.

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Rb (Flesken-Nikitin et al., 2003). Somatic mutations of the p53 gene in sporadic ovarian tumors are common (Milner et al., 1993) and while considerably less is known about the role of Rb gene in ovarian cancer, the two genes do have interconnecting molecular pathways (Sherr and McCormick, 2002). Flesken-Nikitin et al. utilized homozygous transgenic mice that harbored loxP sites flanking a segment of the p53 gene, the Rb gene, or both. To achieve conditional inactivation of these genes limited to the ovarian surface epithelium, AdCre was delivered under the bursal membrane of the ovary. Mice in which both the p53 and Rb genes had been inactivated developed epithelial ovarian tumors at a mean time of 227 days following adenoviral administration. Tumors were mainly classified as either well-differentiated serous neoplasms or poorly differentiated with positive staining for epithelial markers. This model closely mimics the development of epithelial ovarian cancer in women both in terms of the pathology of the tumors, and by the fact that many of the mice developed ascites and metastatic disease in the liver and lungs. The development of this genetically defined model of ovarian cancer will help to answer many questions regarding the early genetic events that take place in the OSE that cause malignant transformation of these cells. The fact that none of the mice in which Rb alone was inactivated and only a very small percentage of those in which p53 alone was inactivated developed ovarian tumors points to a cooperative or perhaps compensatory relationship between the molecular pathways of these two tumor suppressor genes in the OSE. This model will allow researchers to more closely examine the roles of these two genes in the development and progression of ovarian cancer. In the first model using AdCre-mediated gene inactivation, the mice developed epithelial tumors of a serous subtype, which is the most common histopathology of ovarian neoplasms in women (Scully, 1995). There are, however, other histological subtypes, and it has long been shown that mutations of specific genes are associated with these different tumor types. Frequent somatic mutations in the Pten tumor suppressor gene have been observed in a high proportion of endometrioid ovarian tumors, as have mutations in the Kras oncogene, though to a lesser extent (Cuatrecasas et al., 1997; Obata et al., 1998). To examine the effect of aberrant expression of these genes in the OSE, Dinulescu et al. (2005) administered AdCre intrabursally to transgenic mice who bore loxP sites flanking portions of their Pten gene as well as loxP sites surrounding a “stop sequence” preceding the K-ras gene. When either gene was inactivated/activated individually, none of the mice developed neoplasms, save for one floxed Pten mouse. When the expression of both genes was altered concomitantly, all of the mice developed endometrioid ovarian carcinomas with a latency as short as 7 weeks. Perhaps of even greater significance is the fact that in the mice in which the genes were conditionally activated/inactivated individually, the animals developed preneoplastic endometrioid ovarian lesions that closely resembled endometriosis. Thus, while providing valuable insight into

the early genetic events that lead to the development of ovarian tumors with an endometrioid pathology, this study also provided experimental evidence of the long hypothesized link between endometriosis and endometrioid ovarian cancer. In our laboratory, we have generated a line of mice (tgCAG-LS-TAg) that following cre-mediated excision of a “lox-stop” cassette, express the early region of SV40 under the control of the ubiquitous CAG promoter (Fig. 2C). In the absence of cre recombinase, the tgCAG-LS-TAg mice express ␤-galactosidase in a mosaic fashion throughout organs that we have examined. In the ovary, strong ␤-galactosidase expression is evident at what might be recent sites of ovulation (Fig. 2C), based on the presence of underlying corpora lutea. Approximately 30% of tgCAG-LS-TAg mice injected with AdCre proceed to develop epithelial ovarian tumors within 11–25 weeks after AdCre injection. The resulting carcinomas stain positively for the large TAg and CK19. The shorter latency of tumors derived from the activation of TAg as opposed to the inactivation of pRb and p53 (FleskenNikitin et al., 2003) suggests that the early region of SV40 plays a greater role in the malignant transformation of OSE than the simple inactivation of these two tumor suppressors. The Cre–loxP conditional activation/inactivation system has allowed for the development of mouse models of ovarian cancer in which alterations in expression of specific genes can be achieved in a highly tissue-specific and inducible manner, providing a greater level of experimental control than traditional knockout or transgenic models. Unfortunately, the requirement for surgeries and viral preparations of AdCre make it a fairly costly and labor-intensive model. The discovery of a truly ovarian epithelial specific promoter or a targeting strategy for this tissue would not only enable the expression of oncogenes in this tissue, but would more simply permit the expression of cre recombinase in the surface epithelium, foregoing the need for the surgical delivery of AdCre vectors. The development of transgenic mice predisposed to develop ovarian cancer provides an opportunity to model human ovarian cancer in an immune-competent host. Orsulic et al. (2002) noted differences in their model depending on the immune status of the host. The immune system may also play a role in the response of tumors to various therapeutics. As discussed previously, however, the species differences between mouse and human must also be noted when interpreting data from mouse transgenic models. While it might not be possible at present to “humanize” entire signalling pathways in mice, when particular gene products are targeted by therapeutics, the use of the human gene in the transgenic model might eliminate any species differences at the level of therapeutic/protein interaction.

6. Are we there yet? During the past 5 years, the greatest advances in ovarian cancer models have been the generation of transgenic mouse

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models, the development of genetically defined models in murine and human OSE cultures, and the observation that inactivation/activation of different signalling pathways can lead to the emergence of ovarian cancers with different phenotypes and histologies. Exciting future prospects include the detailed examination of the role in tumorigenesis of relevant signalling pathways activated in human ovarian cancer cell lines using RNA interference technology, the further development of transgenic and OSE culture models with an emphasis on the roles of oncogenes and tumor suppressors known to be important in the etiology of human ovarian cancer, and the modeling of ovarian cancers of the different histological subtypes. The most significant challenge for model development remains the identification of a promoter capable of driving gene expression in an OSE cell-specific manner, although an inducible version of the MISIIR promoter may turn out to be suitable. The poor prognosis facing many patients diagnosed with ovarian cancer makes it imperative that early markers for this disease and new therapies and therapeutic targets are identified. The advancement of ovarian cancer models, in addition to providing a means for the pre-screening of therapeutics, holds the promise of developing an improved understanding

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of the genetic changes important for this disease, which in turn will help identify markers for the disease, as well as crucial therapeutic targets. The xenograft model of established human ovarian cancer cell lines represents our best model of late stage ovarian cancer strengthened by the recent appreciation that different cell lines may model different histological subtypes of ovarian cancer. The advent of RNA interference strategies will allow us to better understand which gene products or activated signalling pathways are required for their tumorigenic phenotype. Our understanding of oncogenes and tumor suppressors deemed important for the development of human ovarian cancer can be “put to the test” by exploiting these genetic changes to malignantly transform OSE cells in culture or in situ (transgenic models). Rather than merely asking which oncogenes can malignantly transform OSE, the future direction has already been set where we determine what relevant oncogenes/tumor suppressors are important for forming tumors and how does our choice of oncogenes/tumor suppressors effect the phenotype or histology of the resulting tumors. The various models discussed in this review have identified a number of genetic changes that are essential for tumorigenicity of the ovarian surface epithelium. The inactivation

Fig. 3. Experimental models of human ovarian cancer. Models based on human ovarian cancer cell lines or genetically modified HOSE share species-specific similarities with human ovarian cancer, but their tumorigenicity can only be tested in immune deficient animals. MOSE and transgenic mouse models may display species differences with human cancers, however tumorigenesis can be studied in an immune-competent setting. Other strengths and weaknesses and the future directions and applications of the model systems are summarized.

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of p53, common in epithelial ovarian cancer, was achieved in most of the models either through the action of the large T antigen of SV40, the use of p53-null MOSE, or the conditional inactivation of floxed p53 alleles. Similarly, the inactivation of pRb by the large T antigen or its conditional inactivation following administration of AdCre, is also important for several of the models. Orsulic et al. (2002) reported the requirement for inactivation of p53, but not pRb, in their model; however, efficient transformation of their MOSE required the activation of at least two oncogenes. In contrast, conditional inactivation of pRb and p53 in the surface epithelium of the mouse ovary was sufficient to induce tumors, although the long latency suggests a requirement for acquisition of further genetic changes (Flesken-Nikitin et al., 2003). This long latency was considerably shorter following inactivation of pRb and p53 by the expression of T antigen in the tgCAG-LS-TAg transgenic model; however, additional functions of the large T antigen, small t antigen, and 17 k t antigen may have contributed to this finding. In addition to pRb and p53, several of the recent models also explored the role of the Ras pathway in tumorigenesis. Inactivation of Ha-ras in a xenograft model abrogated tumor formation (Yang et al., 2003), and expression of activated ras genes was sufficient to transform immortalized cells to an epithelial tumorigenic phenotype (Kusakari et al., 2003; Liu et al., 2004). Interestingly, the expression of activated k-ras in normal MOSE in vivo resulted in the development of endometrioid lesions (Dinulescu et al., 2005). Cooperation through the inactivation of Pten pushed these lesions to develop into endometrioid tumors. This is in contrast to the poorly differentiated carcinomas that developed following K-ras expression in immortalized human OSE (Liu et al., 2004). Are the different outcomes in these models due to the inactivation of Pten as opposed to pRB and p53 or are the differences because one system uses in vitro cultured cells and the other is in vivo? While the former interpretation is likely correct based on our knowledge of the association of Pten mutations in endometrioid ovarian cancer, this can be tested through the activation of K-ras and concomitant inactivation of either p53 alone or p53 and pRB. The prediction would be that serous adenocarcinomas would develop in this model. The demonstration of different phenotypes emerging from these models provides a glimpse of the future possibilities: through subtle changes in gene expression, we may be able to model all of the various histological subtypes of epithelial ovarian cancers to better understand their unique etiologies and identify more specific therapeutic targets.

7. Summary In this review, we have highlighted recent advances in ovarian cancer models including xenografts of human ovarian cancer cell lines, the genetic manipulation of human, mouse, and rat ovarian surface epithelial cell lines and the development of mouse transgenic models. While it is unlikely

that any of these models will perfectly reflect the human disease, each model comes with both strengths and weaknesses (Fig. 3). The future of ovarian cancer models will be through the refinement of these models with an improved understanding of these strengths and weaknesses and a careful validation of the findings that emerge from each model system.

Acknowledgements The authors thank Olga Collins and Colleen Crane for their expert technical assistance with the histology and immunohistochemistry data presented in this review. We are also grateful to Drs. Lesley Dunfield and Teresa Woodruff for their critical reviews of the article.

References Abdollahi, A., Godwin, A.K., Miller, P.D., Getts, L.A., Schultz, D.C., Taguchi, T., Testa, J.R., Hamilton, T.C., 1997. Identification of a gene containing zinc-finger motifs based on lost expression in malignantly transformed rat ovarian surface epithelial cells. Cancer Res. 57, 2029–2034. Adams, A.T., Auersperg, N., 1981. Transformation of cultured rat ovarian surface epithelial cells by Kirsten murine sarcoma virus. Cancer Res. 41, 2063–2072. American Cancer Society, 2003. Cancer facts and figures 2003. 5008.03. Atlanta, GA. Auersperg, N., Pan, J., Grove, B.D., Peterson, T., Fisher, J., MainesBandiera, S., Somasiri, A., Roskelley, C.D., 1999. E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium. Proc. Natl. Acad. Sci. U.S.A. 96, 6249–6254. Auersperg, N., Siemens, C.H., Myrdal, S.E., 1984. Human ovarian surface epithelium in primary culture. In Vitro 20, 743–755. Auersperg, N., Wong, A.S., Choi, K.C., Kang, S.K., Leung, P.C., 2001. Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr. Rev. 22, 255–288. Behbod, F., Rosen, J.M., 2005. Will cancer stem cells provide new therapeutic targets? Carcinogenesis 26, 703–711. Buick, R.N., Pullano, R., Trent, J.M., 1985. Comparative properties of five human ovarian adenocarcinoma cell lines. Cancer Res. 45, 3668–3676. Burbridge, M.F., Kraus-Berthier, L., Naze, M., Pierre, A., Atassi, G., Guilbaud, N., 1999. Biological and pharmacological characterisation of three models of human ovarian carcinoma established in nude mice: use of the CA125 tumour marker to predict antitumour activity. Int. J. Oncol. 15, 1155–1162. Chatzistamou, I., Schally, A.V., Pafiti, A., Kiaris, H., Koutselini, H., 2002. Expression of growth hormone-releasing hormone in human primary endometrial carcinomas. Eur. J. Endocrinol. 147, 381–386. Chaudhuri, T.R., Mountz, J.M., Rogers, B.E., Partridge, E.E., Zinn, K.R., 2001. Light-based imaging of green fluorescent protein-positive ovarian cancer xenografts during therapy. Gynecol. Oncol. 82, 581–589. Connolly, D.C., Bao, R., Nikitin, A.Y., Stephens, K.C., Poole, T.W., Hua, X., Harris, S.S., Vanderhyden, B.C., Hamilton, T.C., 2003. Female mice chimeric for expression of the simian virus 40 TAg under control of the MISIIR promoter develop epithelial ovarian cancer. Cancer Res. 63, 1389–1397. Cuatrecasas, M., Villanueva, A., Matias-Guiu, X., Prat, J., 1997. K-ras mutations in mucinous ovarian tumors: a clinicopathologic and molecular study of 95 cases. Cancer 79, 1581–1586. Cvetkovic, D., Pisarcik, D., Lee, C., Hamilton, T.C., Abdollahi, A., 2004. Altered expression and loss of heterozygosity of the LOT1 gene in ovarian cancer. Gynecol. Oncol. 95, 449–455.

K. Garson et al. / Molecular and Cellular Endocrinology 239 (2005) 15–26 Davies, B.R., Steele, I.A., Edmondson, R.J., Zwolinski, S.A., Saretzki, G., von, Z.T., O’Hare, M.J., 2003. Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen. Exp. Cell Res. 288, 390–402. Dinulescu, D.M., Ince, T.A., Quade, B.J., Shafer, S.A., Crowley, D., Jacks, T., 2005. Role of K-ras and Pten in the development of mouse models of endometriosis and endometrioid ovarian cancer. Nat. Med. 11, 63–70. Flesken-Nikitin, A., Choi, K.C., Eng, J.P., Shmidt, E.N., Nikitin, A.Y., 2003. Induction of carcinogenesis by concurrent inactivation of p53 and Rb1 in the mouse ovarian surface epithelium. Cancer Res. 63, 3459–3463. Garson, K., Macdonald, E., Dube, M., Bao, R., Hamilton, T.C., Vanderhyden, B.C., 2003. Generation of tumors in transgenic mice expressing the SV40 T antigen under the control of ovarian-specific promoter 1. J. Soc. Gynecol. Invest. 10, 244–250. Godwin, A.K., Testa, J.R., Handel, L.M., Liu, Z., Vanderveer, L.A., Tracey, P.A., Hamilton, T.C., 1992. Spontaneous transformation of rat ovarian surface epithelial cells: association with cytogenetic changes and implications of repeated ovulation in the etiology of ovarian cancer. J. Natl. Cancer Inst. 84, 592–601. Gossmann, A., Helbich, T.H., Mesiano, S., Shames, D.M., Wendland, M.F., Roberts, T.P., Ferrara, N., Jaffe, R.B., Brasch, R.C., 2000. Magnetic resonance imaging in an experimental model of human ovarian cancer demonstrating altered microvascular permeability after inhibition of vascular endothelial growth factor. Am. J. Obstet. Gynecol. 183, 956–963. Gregoire, L., Munkarah, A., Rabah, R., Morris, R.T., Lancaster, W.D., 1998. Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis. In Vitro Cell Dev. Biol. Anim. 34, 636–639. Hahn, W.C., Weinberg, R.A., 2002. Modelling the molecular circuitry of cancer. Nat. Rev. Cancer 2, 331–341. Hamad, N.M., Elconin, J.H., Karnoub, A.E., Bai, W., Rich, J.N., Abraham, R.T., Der, C.J., Counter, C.M., 2002. Distinct requirements for Ras oncogenesis in human versus mouse cells. Genes Dev. 16, 2045–2057. Hoffman, A.G., Burghardt, R.C., Tilley, R., Auersperg, N., 1993. An in vitro model of ovarian epithelial carcinogenesis: changes in cell-cell communication and adhesion occuring during neoplastic progression. Int. J. Cancer 54, 828–838. Kido, M., Shibuya, M., 1998. Isolation and characterization of mouse ovarian surface epithelial cell lines. Pathol. Res. Pract. 194, 725– 730. Kraeber-Bodere, F., Mishra, A., Thedrez, P., Faivre-Chauvet, A., Bardies, M., Imai, S., Le Boterff, J., Chatal, J.F., 1996. Pharmacokinetics and biodistribution of samarium-153-labelled OC125 antibody coupled to CITCDTPA in a xenograft model of ovarian cancer. Eur. J. Nucl. Med. 23, 560–567. Kusakari, T., Kariya, M., Mandai, M., Tsuruta, Y., Hamid, A.A., Fukuhara, K., Nanbu, K., Takakura, K., Fujii, S., 2003. C-erbB-2 or mutant Ha-ras induced malignant transformation of immortalized human ovarian surface epithelial cells in vitro. Br. J. Cancer 89, 2293–2298. Liu, J., Yang, G., Thompson-Lanza, J.A., Glassman, A., Hayes, K., Patterson, A., Marquez, R.T., Auersperg, N., Yu, Y., Hahn, W.C., Mills, G.B., Bast Jr., R.C., 2004. A genetically defined model for human ovarian cancer. Cancer Res. 64, 1655–1663. Masiakos, P.T., MacLaughlin, D.T., Maheswaran, S., Teixeira, J., Fuller Jr., A.F., Shah, P.C., Kehas, D.J., Kenneally, M.K., Dombkowski, D.M., Ha, T.U., Preffer, F.I., Donahoe, P.K., 1999. Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS. Clin. Cancer Res. 5, 3488– 3499. Meuwissen, R., Jonkers, J., Berns, A., 2001. Mouse models for sporadic cancer. Exp. Cell Res. 264, 100–110.

25

Milner, B.J., Allan, L.A., Eccles, D.M., Kitchener, H.C., Leonard, R.C., Kelly, K.F., Parkin, D.E., Haites, N.E., 1993. p53 Mutation is a common genetic event in ovarian carcinoma. Cancer Res. 53, 2128– 2132. Mobus, V., Gerharz, C.D., Press, U., Moll, R., Beck, T., Mellin, W., Pollow, K., Knapstein, P.G., Kreienberg, R., 1992. Morphological, immunohistochemical and biochemical characterization of 6 newly established human ovarian carcinoma cell lines. Int. J. Cancer 52, 76–84. Molthoff, C.F., Calame, J.J., Pinedo, H.M., Boven, E., 1991. Human ovarian cancer xenografts in nude mice: characterization and analysis of antigen expression. Int. J. Cancer 47, 72–79. Nilsson, E.E., Westfall, S.D., McDonald, C., Lison, T., Sadler-Riggleman, I., Skinner, M.K., 2002. An in vivo mouse reporter gene (human secreted alkaline phosphatase) model to monitor ovarian tumor growth and response to therapeutics. Cancer Chemother. Pharmacol. 49, 93–100. Nitta, M., Katabuchi, H., Ohtake, H., Tashiro, H., Yamaizumi, M., Okamura, H., 2001. Characterization and tumorigenicity of human ovarian surface epithelial cells immortalized by SV40 large T antigen. Gynecol. Oncol. 81, 10–17. Obata, K., Morland, S.J., Watson, R.H., Hitchcock, A., Chenevix-Trench, G., Thomas, E.J., Campbell, I.G., 1998. Frequent PTEN/MMAC mutations in endometrioid but not serous or mucinous epithelial ovarian tumors. Cancer Res. 58, 2095–2097. Orsulic, S., Li, Y., Soslow, R.A., Vitale-Cross, L.A., Gutkind, J.S., Varmus, H.E., 2002. Induction of ovarian cancer by defined multiple genetic changes in a mouse model system. Cancer Cell 1, 53– 62. Roby, K.F., Taylor, C.C., Sweetwood, J.P., Cheng, Y., Pace, J.L., Tawfik, O., Persons, D.L., Smith, P.G., Terranova, P.F., 2000. Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogenesis 21, 585–591. Rygaard, J., Povlsen, C.O., 1969. Heterotransplantation of a human malignant tumour to “Nude” mice. Acta Pathol. Microbiol. Scand. 77, 758–760. Sauer, B., 1998. Inducible gene targeting in mice using the Cre/lox system. Methods 14, 381–392. Scully, R.E., 1995. Pathology of ovarian cancer precursors. J. Cell Biochem. 23 (Suppl.), 208–218. Selvakumaran, M., Bao, R., Crijns, A.P., Connolly, D.C., Weinstein, J.K., Hamilton, T.C., 2001. Ovarian epithelial cell lineage-specific gene expression using the promoter of a retrovirus-like element. Cancer Res. 61, 1291–1295. Shaw, T.J., Senterman, M.K., Dawson, K., Crane, C.A., Vanderhyden, B.C., 2004. Characterization of intraperitoneal, orthotopic, and metastatic xenograft models of human ovarian cancer. Mol. Ther. 10, 1032–1042. Sherr, C.J., McCormick, F., 2002. The RB and p53 pathways in cancer. Cancer Cell 2, 103–112. Sullivan, C.S., Pipas, J.M., 2002. T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis. Microbiol. Mol. Biol. Rev. 66, 179–202. Testa, J.R., Getts, L.A., Salazar, H., Liu, Z., Handel, L.M., Godwin, A.K., Hamilton, T.C., 1994. Spontaneous transformation of rat ovarian surface epithelial cells results in well to poorly differentiated tumors with a parallel range of cytogenetic complexity. Cancer Res. 54, 2778–2784. Tsao, S.W., Mok, S.C., Fey, E.G., Fletcher, J.A., Wan, T.S., Chew, E.C., Muto, M.G., Knapp, R.C., Berkowitz, R.S., 1995. Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPV-E6E7 ORFs). Exp. Cell Res. 218, 499–507. Tsukagoshi, S., Saga, Y., Suzuki, N., Fujioka, A., Nakagawa, F., Fukushima, M., Suzuki, M., 2003. Thymidine phosphorylase-mediated angiogenesis regulated by thymidine phosphorylase inhibitor in human ovarian cancer cells in vivo. Int. J. Oncol. 22, 961–967.

26

K. Garson et al. / Molecular and Cellular Endocrinology 239 (2005) 15–26

Xu, X., Wagner, K.U., Larson, D., Weaver, Z., Li, C., Ried, T., Hennighausen, L., Wynshaw-Boris, A., Deng, C.X., 1999. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat. Genet. 22, 37–43. Yang, G., Thompson, J.A., Fang, B., Liu, J., 2003. Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transforma-

tion efficiency and tumor growth in a model of human ovarian cancer. Oncogene 22, 5694–5701. Zorn, K.K., Jazaeri, A.A., Awtrey, C.S., Gardner, G.J., Mok, S.C., Boyd, J., Birrer, M.J., 2003. Choice of normal ovarian control influences determination of differentially expressed genes in ovarian cancer expression profiling studies. Clin. Cancer Res. 9, 4811–4818.