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Models for human cervical cancer
Vol. 2, No. 1 2005
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Cancer
Models for human cervical cancer Paul F. Lambert*, Tiffany Brake McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Ave., Madison, WI 53706, USA
Cervical cancer, a major world health problem leading to over 200,000 deaths each year, is caused by human papillomaviruses (HPVs). This viral etiology provides a unique opportunity for developing HPV-targeted drugs for preventing or treating cervical cancer. Herein are
Section Editors: Vincent Goffin – INSERM, Faculte´ de Me´decine Necker, Paris, France Christopher J. Ormandy – Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
described model systems for the study of human cervical cancer, with emphasis placed on mouse models that provide potentially important platforms for preclinical studies. Introduction Cervical cancer is a leading cause of death by cancer among women worldwide, with approximately half a million new cases and over 200,000 deaths each year [1]. The Pap smear is a very effective screening tool for cervical cancer; however, women in many countries do not have adequate access to regular testing. Consequently 80% of cervical cancer arises in these developing countries without effective screening [1]. Patients with high grade cervical cancer have extremely poor survival rates, reflective in the high numbers of deaths among those afflicted with this cancer (the worldwide ratio of deaths to incidence is 49%).
Etiology Cervical cancer is caused by sexually transmitted human papillomaviruses (HPV). These HPVs are among the most common sexually transmitted pathogen known today; it is estimated that 75% of sexually active individuals have been infected [2]. In the US over 20 million people are actively infected at any one time with over 5 million new infections arising each year [3]. HPV DNA is found virtually in all cervical cancers. Although there are over a hundred different *Corresponding author: P.F. Lambert ([email protected]) 1740-6757/$ ß 2005 Published by Elsevier Ltd.
DOI: 10.1016/j.ddmod.2005.05.021
HPV genotypes, only thirteen HPV are classified by the World Health Organization as carcinogenic [4]. These HPVs are referred to as the ‘high-risk’ HPVs. Most notable is HPV16, which is found in over half of cervical cancers. High-risk HPVs are also casually associated with other anogenital cancers (e.g. of the vulva, vagina, penis, anus and periungal region) and approximately 20–30% of head and neck cancers, particularly of the tongue, tonsil and oropharynx. Other, cutaneous HPVs have been implicated in squamous cell carcinomas of the skin [4]. The growing recognition that HPVs cause a large number of epithelial cancers raises the opportunity for developing anticancer therapies that target the unique viral etiology of these cancers. The advent of prophylactic HPV vaccines that can prevent infections by certain HPVs holds great promise for reducing the incidence of cervical cancer [5–7]. However, the WHO estimates that these vaccines, currently in clinical trials, will not lead to any discernable reduction in the worldwide incidence of cervical cancer or related deaths until 2040. Furthermore, these vaccines will only protect against two of the thirteen carcinogenic HPVs. Given these facts and because the long-term effectiveness of the vaccines is not known, there remains a strong need to develop more effective treatments for cervical and other HPV-associated cancers. In HPV-associated cancers, two viral oncogenes, E6 and E7, are commonly found to be upregulated in their expression as a consequence of the viral genome becoming integrated into the host chromosome. The selective expression of E6 and E7 in HPV-associated cancer led to the prediction that they contribute to this cancer. Cervical epithelial cells harbouring www.drugdiscoverytoday.com
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integrated HPV-16 DNA have a selective growth advantage over cells harbouring normal extrachromosomal viral genomes; and this growth advantage correlates with the increased expression of two viral genes in particular, E6 and E7 [8]. E6 and E7 bind and inactivate the tumour suppressor gene products, p53 and pRB, respectively [9,10]. In cell lines derived from HPV-positive cervical cancers these cellular genes are not mutationally inactivated; however, they are mutated in HPV-negative cervical cancer-derived cell lines [11]. The expression of the E6 and E7 viral genes is required for the continued growth of cervical cancerderived cell lines [12]. These facts support the hypothesis that E6 and E7 are causally related to the onset and/or maintenance of human cervical cancers. Numerous studies including the ones described herein have provided overwhelming evidence that the high-risk HPV E6 and E7 genes encode multifunctional proteins that confer oncogenic properties in tissue culture and in vivo. Cervical cancer is neither the immediate, nor the necessary, result of high-risk HPV infection. These cancers usually take over two decades to arise following exposure to these viruses. Cancer only develops in a fraction of the infected women. These facts imply that HPV is not the only factor that contributes to the development of cervical cancer. Epidemiological studies have identified tobacco smoke, hormones, parity, genetic factors, immune deficiencies and immune suppression as additional factors contributing to cervical cancer in addition to HPVs [4]. Much of our current understanding of the role of HPVs in cervical cancer arose from in vitro studies in tissue culture. Using these in vitro model systems, a number of biological properties of E6 and E7 were identified that probably reflect their oncogenic potential. Below, we briefly describe the insights gained from these in vitro studies, which have been reviewed at length elsewhere [13]. Of greater relevance to drug discovery efforts, HPV-transgenic animal models for human cervical cancer have now been developed. These animal model systems not only provide the means for determining the relative contributions of these different factors in human cervical cancer but also provide a preclinical screening platform for identifying novel drugs for the treatment or prevention of cervical cancer. In this contribution, we focus on describing mouse models for the study of human cervical cancer, their validation, the insights gained from their study, their value in preclinical screening and future directions of research regarding the development and use of such models.
In vitro models Several biological properties have been ascribed to HPVs in tissue culture model systems that provide readouts for the oncogenic potential of these viruses, and in particular the E6 and E7 oncogenes. Below are described three major biological properties that reflect the oncogenic potential of E6 and E7: 16
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immortalization, genomic instability and modulation of DNA damage responses. For each property, insight is provided into the mechanism of action by which E6 and E7 confer this property to cells in culture. The E6 and E7 genes from the high-risk HPVs are transforming in tissue culture. E7 cooperates with an activated ras to transform baby rat kidney or human cervical epithelial cells [14–16]. E6 and E7 can act independently or synergistically to immortalize multiple cell types including human foreskin keratinocytes, cervical epithelial, or mammary epithelial cells [17–22]. The ability of HPV16 E7 to contribute to immortalization correlates with its disruption of the p16/ pRb pathway [23]. The mechanism by which E6 contributes to immortalization is controversial. Some investigators have correlated its immortalization potential to its ability to induce expression of telomerase [23]. Telomerase activity clearly is induced by E6 [24]. The level of telomerase activity in E6-positive cells increases further at the time cells become immortalized even though levels of E6 expression do not change [25], arguing for other events contributing to telomerase activation. Other investigators have linked E6’s immortalization potential in mammary epithelial and in keratinocytes to its inactivation of p53 [26,27]. Another hallmark of E6- and E7-expressing cells is genomic instability, which has been observed in multiple epithelial cell types [28,29]. Mitotic abnormalities can be induced by HPV 16 E6 and E7 oncoproteins by direct subversion of the mitotic spindle checkpoint [30,31]. The ability of E6 to induce genomic instability probably reflects its ability to inhibit p53 function [32], leading to the disruption of normal DNA repair processes and the consequent accumulation of genetic change. In the case of E7, genomic instability might reflect its effect on centrosome biogenesis, and the consequent defects in segregation of daughter chromosomes during cell division [33–35]. Both E6 and E7 can abrogate normal DNA damage responses [36–38]. This is thought to reflect an ability of E6 and E7 each to block the p53 mediated cell cycle arrest. In the case of E6 this correlates at least in part with the ability of E6 to bind and inactivate p53 [39]. For E7, this property correlates not only with its ability to disrupt the function of the cell cycle regulator pRb, but also with its ability to inactivate p21 [40,41], the cyclin-dependent kinase inhibitor that is induced in its expression when p53 is activated in response to DNA damage [42]. Abrogation of the DNA damage responses is hypothesized to contribute to the accumulation of genetic alterations in HPV-positive cells including those, which might contribute to the oncogenicity.
In vivo models The first mouse model for HPV-induced cervical cancer was one, which employed the use of a recombinant retrovirus to transduce the HPV16 E6 and E7 oncogenes into the mouse
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Drug Discovery Today: Disease Models | Cancer
in the cervix compared to E6 in the skin [46,47]. Specifically, treatment of nontransgenic mice with estrogen leads to epithelial hyperplasia with an absence of neoplasia (Fig. 2). Treatment of E6 transgenic mice led to a similar phenotype. Treatment of E7 mice led to neoplastic conversion of the cervical epithelium, which manifests as highly dyplastic cervical intraepithelial neoplasia grade III (CINIII)-like lesions, carcinoma in situ (CIS), and microinvasive squamous carcinoma, as seen in women with progressive cervical disease (Fig. 2). Treatment of E6 E7 doubly transgenic mice with estrogen led to large invasive carcinoma (Fig. 2).
Model validation
Figure 1. Mouse model for cervical cancer. Shown is a schematic representation of the cervical cancer mouse model system. As described in Riley et al. [46], female HPV transgenic mice are given slow release pellets containing 17b-estradiol for a period of six months, after which reproductive tracts are harvested for histological analysis. Every tenth 5 uM section is stained with hematoxylin and eosin and histopathologically evaluated. The box demarks the location of the cervix within the reproductive tract.
cervix [43]. Cervical cancer developed when the mice exposed to the E6/E7 recombinant retrovirus were treated with TPA (phorbol-13-myristate-12-acetate, a tumor promoter) or MNNG (N-methyl-N0 -nitro-N-nitrosoguanidine, a mutagen). This mouse model was not very reliable and was very labor-intensive. A more tractable model for HPVinduced carcinogenesis was developed in which K14HPV16 transgenic mice were treated with exogenous estrogen (Fig. 1). These mice developed a progressive disease leading to the formation of squamous carcinoma of the cervix over a six-month period that closely reflects the histopathological characteristics of the progressive disease leading to the cervical cancer in humans [44,45]. In addition squamous carcinoma was observed frequently in the vagina, an organ for which HPVs association with cancer has also been epidemiologically established [4]. The individual roles of E6 and E7 in cervical cancer were found to differ from that previously observed in the skin with E7 playing a more dominant role
The validity of these HPV16 transgenic mouse models as models for human cervical cancer has been demonstrated at multiple levels. First the histopathological progression of the disease in the mouse closely parallels that in human cervical cancer, with hyperplasia giving rise to low-grade (CIN I) to high-grade (CIN III) dysplasia, and ultimately frank cancer [46]. Second, the role of estrogen as a cofactor in the development and the progression of cervical cancers in the HPV transgenic mice [48] parallels the epidemiological evidence for a role of estrogens in human cancer inferred from the increased risk for cervical cancer among women using oral contraceptives for long periods [49], those with high parity, or both [50]. And third, there is a close parallel in the observed pattern of expression of biomarkers for human and mouse cervical cancer; furthermore, the mouse model has been predictive in identifying MCM7 as an informative new biomarker for human cervical cancer [51].
Use mouse models to evaluate immunological responses to HPV oncoproteins and develop effective immunotherapies against cervical cancer With the generation of HPV transgenic mouse models expressing viral antigens arose the opportunity to investigate immune recognition of these antigens. The K14E6 and K14E7 mice, used in the above-described cervical cancer studies, provide such an opportunity. These mice express the E6 or E7 oncogene not only in the epithelial lining of the cervix, but also in the epidermis lining the skin [52,53], affording one with the ability of monitoring immune recognition of the viral oncoproteins in the context of skin grafting studies, in which the skin of the transgenic mice is grafted onto syngeneic, nontransgenic littermates of the same inbred genetic background. Surprisingly, E7 was found not to function as a classical minor transplantation antigen in the context of its expression in mouse epidermis. Skin from K14E7 mice when grafted onto nontransgenic mice was not rejected even when these mice were immunized against E7 and developed E7-specific CTL responses [54]. Similarly, grafts in which E6 antigen is expressed from the K14 promoter are also accepted [55]. However, recipient animals could be induced www.drugdiscoverytoday.com
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Figure 2. Histopathology of cervix in estrogen-treated mice. Shown are representative high magnification microscopic images from the cervix of estrogen-treated nontransgenic (panel (a)), K14E6 transgenic (panel (b)), and K14E7 transgenic (panel (c)) mice. Note the hyperplasia of the cervical epithelia in panels (a) and (b), and versus the presence of CINIII/CIS in panel (c). Shown in panel (d) is a low magnification image of the cervix of estrogentreated K14E6 K14E7 transgenic mice showing the presence of a large tumor involving the entire cervix. Inset in lower right corner shows high magnification image demonstrating the presence of highly invasive squamous cell carcinoma within this tumor mass.
to reject the skin grafts in an E7-specific manner by stimulating systemic proinflammatory responses [54], or through passive transfer of E7-specific CTLs in combination with E7-specific immunization [55]. The insights gained from these grafting studies have clear implications regarding the design of effective therapeutic vaccines for treating patients with HPV-associated disease.
Comparing the value of autochthonous (e.g. HPV transgenic) mouse models over traditional xenograft models Animal experimentation for measuring drug efficacy in preventing cancers has traditionally relied upon the use of xenograft models in which human tumor-derived cell lines, 18
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established and passaged in tissue culture, are implanted subcutaneously into immuno-deficient strains of mice and their ability to form tumors scored in the presence or absence of drug. Although straightforward to use, the relevance of these models can be questioned. The tumors that arise in these xenograft models do not do so in the context of the natural organ environment, and therefore lack the normal interactions that tumors must establish with neighboring normal cells and stroma. In addition, the xenograft models are susceptible to changes in tumor cell character, which arise from their having been selected for growth in tissue culture and passaged over time. These changes could influence their response to drugs. There is a growing appreciation that xenograft models do not necessarily provide reliable indica-
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tors for drug efficacy [56,57]. More recently developed autochthonous mouse models (ACMs) for human cancer, such as the HPV transgenic mouse models for human cervical cancer described herein, are probably to provide more reliable models for testing the efficacy of drugs in treating human cancers because they are not subject to the two caveats raised above for the xenograft models [58,59]. In ACMs, tumors arise within the context of the natural organ site (therefore are subjected to the analogous microenvironment as in human cancers) and they are not subject to the potential influences placed on cells grown in tissue culture. There have been encouraging results in cases where ACMs have been evaluated for drug responsiveness to drugs with known efficacy in preventing or treating analogous human cancers. For example, the responsiveness of myeloid leukemias to retinoic acid and arsenic has been evaluated in ACMs and found to compare well to the properties of the analogous human cancer [60,61]. Similarly, the Apcmin mouse model for colon cancer has demonstrated to respond favorably to COX2 inhibitors in a similar manner to that observed with human families carrying a mutant allele in Apc, which are predisposed to intestinal cancers [62]. Of direct relevance to the HPV transgenic mouse model for human cervical cancer are the successful preclinical studies that demonstrated the efficacy of indole-3-carbinol in preventing cervical cancer in these mice [63,64]. This led to a clinical trial in which indole-3-carbinol was found to be highly effective in causing an increased rate of regression of CIN III in women [65].
Conclusions HPV transgenic mice provide a tractable in vivo preclinical model for human cervical cancer. The validity of these mouse models has been established, and their value demonstrated by the identification of a new biomarker for human cervical cancer, the characterization of estrogen as a potential target for anticancer treatment, and the demonstration of the effectiveness of indole-3-carbinol in preventing cervical neoplasia both in the mouse model and in women.
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Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Cancer
Models for human cervical cancer Paul F. Lambert*, Tiffany Brake McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Ave., Madison, WI 53706, USA
Cervical cancer, a major world health problem leading to over 200,000 deaths each year, is caused by human papillomaviruses (HPVs). This viral etiology provides a unique opportunity for developing HPV-targeted drugs for preventing or treating cervical cancer. Herein are
Section Editors: Vincent Goffin – INSERM, Faculte´ de Me´decine Necker, Paris, France Christopher J. Ormandy – Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
described model systems for the study of human cervical cancer, with emphasis placed on mouse models that provide potentially important platforms for preclinical studies. Introduction Cervical cancer is a leading cause of death by cancer among women worldwide, with approximately half a million new cases and over 200,000 deaths each year [1]. The Pap smear is a very effective screening tool for cervical cancer; however, women in many countries do not have adequate access to regular testing. Consequently 80% of cervical cancer arises in these developing countries without effective screening [1]. Patients with high grade cervical cancer have extremely poor survival rates, reflective in the high numbers of deaths among those afflicted with this cancer (the worldwide ratio of deaths to incidence is 49%).
Etiology Cervical cancer is caused by sexually transmitted human papillomaviruses (HPV). These HPVs are among the most common sexually transmitted pathogen known today; it is estimated that 75% of sexually active individuals have been infected [2]. In the US over 20 million people are actively infected at any one time with over 5 million new infections arising each year [3]. HPV DNA is found virtually in all cervical cancers. Although there are over a hundred different *Corresponding author: P.F. Lambert ([email protected]) 1740-6757/$ ß 2005 Published by Elsevier Ltd.
DOI: 10.1016/j.ddmod.2005.05.021
HPV genotypes, only thirteen HPV are classified by the World Health Organization as carcinogenic [4]. These HPVs are referred to as the ‘high-risk’ HPVs. Most notable is HPV16, which is found in over half of cervical cancers. High-risk HPVs are also casually associated with other anogenital cancers (e.g. of the vulva, vagina, penis, anus and periungal region) and approximately 20–30% of head and neck cancers, particularly of the tongue, tonsil and oropharynx. Other, cutaneous HPVs have been implicated in squamous cell carcinomas of the skin [4]. The growing recognition that HPVs cause a large number of epithelial cancers raises the opportunity for developing anticancer therapies that target the unique viral etiology of these cancers. The advent of prophylactic HPV vaccines that can prevent infections by certain HPVs holds great promise for reducing the incidence of cervical cancer [5–7]. However, the WHO estimates that these vaccines, currently in clinical trials, will not lead to any discernable reduction in the worldwide incidence of cervical cancer or related deaths until 2040. Furthermore, these vaccines will only protect against two of the thirteen carcinogenic HPVs. Given these facts and because the long-term effectiveness of the vaccines is not known, there remains a strong need to develop more effective treatments for cervical and other HPV-associated cancers. In HPV-associated cancers, two viral oncogenes, E6 and E7, are commonly found to be upregulated in their expression as a consequence of the viral genome becoming integrated into the host chromosome. The selective expression of E6 and E7 in HPV-associated cancer led to the prediction that they contribute to this cancer. Cervical epithelial cells harbouring www.drugdiscoverytoday.com
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integrated HPV-16 DNA have a selective growth advantage over cells harbouring normal extrachromosomal viral genomes; and this growth advantage correlates with the increased expression of two viral genes in particular, E6 and E7 [8]. E6 and E7 bind and inactivate the tumour suppressor gene products, p53 and pRB, respectively [9,10]. In cell lines derived from HPV-positive cervical cancers these cellular genes are not mutationally inactivated; however, they are mutated in HPV-negative cervical cancer-derived cell lines [11]. The expression of the E6 and E7 viral genes is required for the continued growth of cervical cancerderived cell lines [12]. These facts support the hypothesis that E6 and E7 are causally related to the onset and/or maintenance of human cervical cancers. Numerous studies including the ones described herein have provided overwhelming evidence that the high-risk HPV E6 and E7 genes encode multifunctional proteins that confer oncogenic properties in tissue culture and in vivo. Cervical cancer is neither the immediate, nor the necessary, result of high-risk HPV infection. These cancers usually take over two decades to arise following exposure to these viruses. Cancer only develops in a fraction of the infected women. These facts imply that HPV is not the only factor that contributes to the development of cervical cancer. Epidemiological studies have identified tobacco smoke, hormones, parity, genetic factors, immune deficiencies and immune suppression as additional factors contributing to cervical cancer in addition to HPVs [4]. Much of our current understanding of the role of HPVs in cervical cancer arose from in vitro studies in tissue culture. Using these in vitro model systems, a number of biological properties of E6 and E7 were identified that probably reflect their oncogenic potential. Below, we briefly describe the insights gained from these in vitro studies, which have been reviewed at length elsewhere [13]. Of greater relevance to drug discovery efforts, HPV-transgenic animal models for human cervical cancer have now been developed. These animal model systems not only provide the means for determining the relative contributions of these different factors in human cervical cancer but also provide a preclinical screening platform for identifying novel drugs for the treatment or prevention of cervical cancer. In this contribution, we focus on describing mouse models for the study of human cervical cancer, their validation, the insights gained from their study, their value in preclinical screening and future directions of research regarding the development and use of such models.
In vitro models Several biological properties have been ascribed to HPVs in tissue culture model systems that provide readouts for the oncogenic potential of these viruses, and in particular the E6 and E7 oncogenes. Below are described three major biological properties that reflect the oncogenic potential of E6 and E7: 16
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immortalization, genomic instability and modulation of DNA damage responses. For each property, insight is provided into the mechanism of action by which E6 and E7 confer this property to cells in culture. The E6 and E7 genes from the high-risk HPVs are transforming in tissue culture. E7 cooperates with an activated ras to transform baby rat kidney or human cervical epithelial cells [14–16]. E6 and E7 can act independently or synergistically to immortalize multiple cell types including human foreskin keratinocytes, cervical epithelial, or mammary epithelial cells [17–22]. The ability of HPV16 E7 to contribute to immortalization correlates with its disruption of the p16/ pRb pathway [23]. The mechanism by which E6 contributes to immortalization is controversial. Some investigators have correlated its immortalization potential to its ability to induce expression of telomerase [23]. Telomerase activity clearly is induced by E6 [24]. The level of telomerase activity in E6-positive cells increases further at the time cells become immortalized even though levels of E6 expression do not change [25], arguing for other events contributing to telomerase activation. Other investigators have linked E6’s immortalization potential in mammary epithelial and in keratinocytes to its inactivation of p53 [26,27]. Another hallmark of E6- and E7-expressing cells is genomic instability, which has been observed in multiple epithelial cell types [28,29]. Mitotic abnormalities can be induced by HPV 16 E6 and E7 oncoproteins by direct subversion of the mitotic spindle checkpoint [30,31]. The ability of E6 to induce genomic instability probably reflects its ability to inhibit p53 function [32], leading to the disruption of normal DNA repair processes and the consequent accumulation of genetic change. In the case of E7, genomic instability might reflect its effect on centrosome biogenesis, and the consequent defects in segregation of daughter chromosomes during cell division [33–35]. Both E6 and E7 can abrogate normal DNA damage responses [36–38]. This is thought to reflect an ability of E6 and E7 each to block the p53 mediated cell cycle arrest. In the case of E6 this correlates at least in part with the ability of E6 to bind and inactivate p53 [39]. For E7, this property correlates not only with its ability to disrupt the function of the cell cycle regulator pRb, but also with its ability to inactivate p21 [40,41], the cyclin-dependent kinase inhibitor that is induced in its expression when p53 is activated in response to DNA damage [42]. Abrogation of the DNA damage responses is hypothesized to contribute to the accumulation of genetic alterations in HPV-positive cells including those, which might contribute to the oncogenicity.
In vivo models The first mouse model for HPV-induced cervical cancer was one, which employed the use of a recombinant retrovirus to transduce the HPV16 E6 and E7 oncogenes into the mouse
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in the cervix compared to E6 in the skin [46,47]. Specifically, treatment of nontransgenic mice with estrogen leads to epithelial hyperplasia with an absence of neoplasia (Fig. 2). Treatment of E6 transgenic mice led to a similar phenotype. Treatment of E7 mice led to neoplastic conversion of the cervical epithelium, which manifests as highly dyplastic cervical intraepithelial neoplasia grade III (CINIII)-like lesions, carcinoma in situ (CIS), and microinvasive squamous carcinoma, as seen in women with progressive cervical disease (Fig. 2). Treatment of E6 E7 doubly transgenic mice with estrogen led to large invasive carcinoma (Fig. 2).
Model validation
Figure 1. Mouse model for cervical cancer. Shown is a schematic representation of the cervical cancer mouse model system. As described in Riley et al. [46], female HPV transgenic mice are given slow release pellets containing 17b-estradiol for a period of six months, after which reproductive tracts are harvested for histological analysis. Every tenth 5 uM section is stained with hematoxylin and eosin and histopathologically evaluated. The box demarks the location of the cervix within the reproductive tract.
cervix [43]. Cervical cancer developed when the mice exposed to the E6/E7 recombinant retrovirus were treated with TPA (phorbol-13-myristate-12-acetate, a tumor promoter) or MNNG (N-methyl-N0 -nitro-N-nitrosoguanidine, a mutagen). This mouse model was not very reliable and was very labor-intensive. A more tractable model for HPVinduced carcinogenesis was developed in which K14HPV16 transgenic mice were treated with exogenous estrogen (Fig. 1). These mice developed a progressive disease leading to the formation of squamous carcinoma of the cervix over a six-month period that closely reflects the histopathological characteristics of the progressive disease leading to the cervical cancer in humans [44,45]. In addition squamous carcinoma was observed frequently in the vagina, an organ for which HPVs association with cancer has also been epidemiologically established [4]. The individual roles of E6 and E7 in cervical cancer were found to differ from that previously observed in the skin with E7 playing a more dominant role
The validity of these HPV16 transgenic mouse models as models for human cervical cancer has been demonstrated at multiple levels. First the histopathological progression of the disease in the mouse closely parallels that in human cervical cancer, with hyperplasia giving rise to low-grade (CIN I) to high-grade (CIN III) dysplasia, and ultimately frank cancer [46]. Second, the role of estrogen as a cofactor in the development and the progression of cervical cancers in the HPV transgenic mice [48] parallels the epidemiological evidence for a role of estrogens in human cancer inferred from the increased risk for cervical cancer among women using oral contraceptives for long periods [49], those with high parity, or both [50]. And third, there is a close parallel in the observed pattern of expression of biomarkers for human and mouse cervical cancer; furthermore, the mouse model has been predictive in identifying MCM7 as an informative new biomarker for human cervical cancer [51].
Use mouse models to evaluate immunological responses to HPV oncoproteins and develop effective immunotherapies against cervical cancer With the generation of HPV transgenic mouse models expressing viral antigens arose the opportunity to investigate immune recognition of these antigens. The K14E6 and K14E7 mice, used in the above-described cervical cancer studies, provide such an opportunity. These mice express the E6 or E7 oncogene not only in the epithelial lining of the cervix, but also in the epidermis lining the skin [52,53], affording one with the ability of monitoring immune recognition of the viral oncoproteins in the context of skin grafting studies, in which the skin of the transgenic mice is grafted onto syngeneic, nontransgenic littermates of the same inbred genetic background. Surprisingly, E7 was found not to function as a classical minor transplantation antigen in the context of its expression in mouse epidermis. Skin from K14E7 mice when grafted onto nontransgenic mice was not rejected even when these mice were immunized against E7 and developed E7-specific CTL responses [54]. Similarly, grafts in which E6 antigen is expressed from the K14 promoter are also accepted [55]. However, recipient animals could be induced www.drugdiscoverytoday.com
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Figure 2. Histopathology of cervix in estrogen-treated mice. Shown are representative high magnification microscopic images from the cervix of estrogen-treated nontransgenic (panel (a)), K14E6 transgenic (panel (b)), and K14E7 transgenic (panel (c)) mice. Note the hyperplasia of the cervical epithelia in panels (a) and (b), and versus the presence of CINIII/CIS in panel (c). Shown in panel (d) is a low magnification image of the cervix of estrogentreated K14E6 K14E7 transgenic mice showing the presence of a large tumor involving the entire cervix. Inset in lower right corner shows high magnification image demonstrating the presence of highly invasive squamous cell carcinoma within this tumor mass.
to reject the skin grafts in an E7-specific manner by stimulating systemic proinflammatory responses [54], or through passive transfer of E7-specific CTLs in combination with E7-specific immunization [55]. The insights gained from these grafting studies have clear implications regarding the design of effective therapeutic vaccines for treating patients with HPV-associated disease.
Comparing the value of autochthonous (e.g. HPV transgenic) mouse models over traditional xenograft models Animal experimentation for measuring drug efficacy in preventing cancers has traditionally relied upon the use of xenograft models in which human tumor-derived cell lines, 18
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established and passaged in tissue culture, are implanted subcutaneously into immuno-deficient strains of mice and their ability to form tumors scored in the presence or absence of drug. Although straightforward to use, the relevance of these models can be questioned. The tumors that arise in these xenograft models do not do so in the context of the natural organ environment, and therefore lack the normal interactions that tumors must establish with neighboring normal cells and stroma. In addition, the xenograft models are susceptible to changes in tumor cell character, which arise from their having been selected for growth in tissue culture and passaged over time. These changes could influence their response to drugs. There is a growing appreciation that xenograft models do not necessarily provide reliable indica-
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tors for drug efficacy [56,57]. More recently developed autochthonous mouse models (ACMs) for human cancer, such as the HPV transgenic mouse models for human cervical cancer described herein, are probably to provide more reliable models for testing the efficacy of drugs in treating human cancers because they are not subject to the two caveats raised above for the xenograft models [58,59]. In ACMs, tumors arise within the context of the natural organ site (therefore are subjected to the analogous microenvironment as in human cancers) and they are not subject to the potential influences placed on cells grown in tissue culture. There have been encouraging results in cases where ACMs have been evaluated for drug responsiveness to drugs with known efficacy in preventing or treating analogous human cancers. For example, the responsiveness of myeloid leukemias to retinoic acid and arsenic has been evaluated in ACMs and found to compare well to the properties of the analogous human cancer [60,61]. Similarly, the Apcmin mouse model for colon cancer has demonstrated to respond favorably to COX2 inhibitors in a similar manner to that observed with human families carrying a mutant allele in Apc, which are predisposed to intestinal cancers [62]. Of direct relevance to the HPV transgenic mouse model for human cervical cancer are the successful preclinical studies that demonstrated the efficacy of indole-3-carbinol in preventing cervical cancer in these mice [63,64]. This led to a clinical trial in which indole-3-carbinol was found to be highly effective in causing an increased rate of regression of CIN III in women [65].
Conclusions HPV transgenic mice provide a tractable in vivo preclinical model for human cervical cancer. The validity of these mouse models has been established, and their value demonstrated by the identification of a new biomarker for human cervical cancer, the characterization of estrogen as a potential target for anticancer treatment, and the demonstration of the effectiveness of indole-3-carbinol in preventing cervical neoplasia both in the mouse model and in women.
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