Impact of p53 loss on reversal and recurrence of conditional Wnt-induced tumorigenesis.
Journal: 2003/March - Genes and Development
ISSN: 0890-9369
Abstract:
Aberrant activation of Wnt signaling is oncogenic and has been implicated in a variety of human cancers. We have developed a doxycycline-inducible Wnt1 transgenic mouse model to determine the dependence of established mammary adenocarcinomas on continued Wnt signaling. Using this model we show that targeted down-regulation of the Wnt pathway results in the rapid disappearance of essentially all Wnt-initiated invasive primary tumors as well as pulmonary metastases. Tumor regression does not require p53 and occurs even in highly aneuploid tumors. However, despite the dependence of primary mammary tumors and metastases on continued Wnt signaling and the dispensability of p53 for tumor regression, we find that a substantial fraction of tumors progress to a Wnt-independent state and that p53 suppresses this process. Specifically, loss of one p53 allele dramatically facilitates the progression of mammary tumors to a Wnt1-independent state both by impairing the regression of primary tumors following doxycycline withdrawal and by promoting the recurrence of fully regressed tumors in the absence of doxycycline. Thus, although p53 itself is dispensable for tumor regression, it nevertheless plays a critical role in the suppression of tumor recurrence. Our findings demonstrate that although even advanced stages of epithelial malignancy remain dependent upon continued Wnt signaling for maintenance and growth, loss of p53 facilitates tumor escape and the acquisition of oncogene independence.
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Genes Dev 17(4): 488-501

Impact of p53 loss on reversal and recurrence of conditional Wnt-induced tumorigenesis

Department of Cancer Biology, Department of Cell and Developmental Biology, Department of Medicine, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA; Center for Comparative Medicine, University of California, Davis, Davis, California 95616, USA
Corresponding author.
Received 2002 Oct 18; Accepted 2002 Dec 31.

Abstract

Aberrant activation of Wnt signaling is oncogenic and has been implicated in a variety of human cancers. We have developed a doxycycline-inducible Wnt1 transgenic mouse model to determine the dependence of established mammary adenocarcinomas on continued Wnt signaling. Using this model we show that targeted down-regulation of the Wnt pathway results in the rapid disappearance of essentially all Wnt-initiated invasive primary tumors as well as pulmonary metastases. Tumor regression does not require p53 and occurs even in highly aneuploid tumors. However, despite the dependence of primary mammary tumors and metastases on continued Wnt signaling and the dispensability of p53 for tumor regression, we find that a substantial fraction of tumors progress to a Wnt-independent state and that p53 suppresses this process. Specifically, loss of one p53 allele dramatically facilitates the progression of mammary tumors to a Wnt1-independent state both by impairing the regression of primary tumors following doxycycline withdrawal and by promoting the recurrence of fully regressed tumors in the absence of doxycycline. Thus, although p53 itself is dispensable for tumor regression, it nevertheless plays a critical role in the suppression of tumor recurrence. Our findings demonstrate that although even advanced stages of epithelial malignancy remain dependent upon continued Wnt signaling for maintenance and growth, loss of p53 facilitates tumor escape and the acquisition of oncogene independence.

Keywords: Wnt, p53, mammary gland, inducible transgenic animals
Abstract

Wnt signaling during development governs cell proliferation and cell fate in multiple tissues from organisms as diverse as flies and mice (Cadigan and Nusse 1997). Consequently, mutations leading to aberrant activation of Wnt signaling can perturb tissue patterning as well as provide an oncogenic stimulus. Indeed, the Wnt pathway was initially discovered as a result of studies of mammary tumorigenesis in the mouse. Specifically, murine tumors arising as a consequence of infection with the mouse mammary tumor virus (MMTV) were frequently found to harbor retroviral insertions in the vicinity of the Wnt1 locus (formerly known as Int-1) that result in activation of Wnt1 expression (Nusse and Varmus 1982). The subsequent demonstration that forced expression of Wnt1 in the mammary glands of transgenic mice results in hyperplasias and adenocarcinomas of the ductal epithelium confirmed that Wnt1 is a proto-oncogene (Tsukamoto et al. 1988). A clear link has also emerged between activation of the Wnt pathway and human cancer in that several genes that regulate Wnt signaling have proven to be either proto-oncogenes, as in the case of β-catenin, or tumor suppressor genes, as in the case of APC and AXIN (Polakis 2000). Consistent with this model, mutations in either Apc or β-catenin predispose mice to mammary ductal hyperplasias and invasive tumors (Moser et al. 1993; Imbert et al. 2001; Michaelson and Leder 2001; Miyoshi et al. 2002). Although downstream targets of Wnt signaling, such as MYC and cyclin D1, are known to play important roles in the pathogenesis of human breast cancer, a causal link between aberrant Wnt signaling and human breast cancer is, as yet, unproved. Nevertheless, there is increasing evidence that expression of components of the Wnt signaling pathway itself are altered in a high percentage of human breast cancers (for review, see Bergstein and Brown 1999; Ugolini et al. 2001; Jonsson et al. 2002).

Although it is clear that activation of canonical Wnt signaling plays a role in the genesis of a variety of human tumors, whether established tumors remain dependent on continued Wnt signaling for maintenance and growth is unknown (Bienz and Clevers 2000; Taipale and Beachy 2001). While it is possible that Wnt-initiated tumors might be susceptible to antineoplastic strategies involving the targeted down-regulation of Wnt signaling, it is also possible that the accumulation of genetic lesions that foster tumor progression may concomitantly render Wnt pathway activation dispensable for tumor cell survival and proliferation. Unfortunately, analyzing spontaneous events that contribute to tumor progression is difficult, if not impossible, using in vitro systems due to the artificial milieu in which these changes occur. In contrast, genetically engineered mice offer tractable in vivo models that can faithfully recapitulate the stepwise progression of human solid tumors (Siegel et al. 2000). For this reason, inducible transgenic mouse models that permit the conditional expression of oncogenes are of particular interest for modeling the effects of targeted therapies, because the abrogation of oncogene expression following the withdrawal of inducer can simulate drug-mediated loss-of-function in a defined oncogenic pathway (Jain et al. 2002). Most notably, the use of a genetic approach to down-regulate oncogenic signaling permits the effects of a targeted therapy to be modeled within an intact tumor composed of multiple cell types in advance of drug development.

To analyze the consequences of down-regulating Wnt signaling in tumors initiated by Wnt pathway activation, we developed a doxycycline-dependent mouse model in which Wnt1 can be conditionally expressed in the mammary epithelium of transgenic mice. Chronic induction of Wnt1 expression in this model results in the stochastic formation of mammary adenocarcinomas over periods of up to a year. In this article we examine the dependence of Wnt1-initiated mammary tumors on continued Wnt signaling in a variety of contexts characteristic of advanced epithelial malignancy. In particular, we tested whether acquisition of a metastatic phenotype, loss of p53 function, or tumor cell aneuploidy preclude tumor regression following abrogation of Wnt1 expression. We demonstrate here that abrogating Wnt1 expression in tumor cells results in the rapid and extensive regression of both primary mammary tumors and pulmonary metastases. These observations provide direct evidence that Wnt pathway down-regulation has potent antineoplastic activity in vivo even against the most advanced stages of epithelial malignancy. In addition, primary tumors lacking p53, including those exhibiting gross aneuploidy, were also found to be capable of undergoing complete regression following the abrogation of Wnt transgene expression. Hence, manifestations of tumor progression such as metastatic spread, chromosomal instability, and loss of p53 do not preclude tumor regression following the targeted down-regulation of an oncogenic signaling pathway. Nevertheless, although our data demonstrate that p53 itself is not required for the regression of Wnt-induced tumors, loss of one p53 allele strongly facilitated the emergence of tumor cells that had progressed to a Wnt1-independent state.

Mammary tumors arising in MTB/TWNT/p53(+/+) and MTB/TWNT/p53(+/−) mice were biopsied, and tumor size was monitored following doxycycline withdrawal. Tumors occurring in p53(+/−) mice were significantly less likely to regress completely following doxycycline withdrawal than tumors occurring in p53(+/+) mice (p < 0.005, chi-square). In addition, tumors that failed to fully regress promptly resumed growth.

Mammary tumors arising in MTB/TWNT/p53(+/−) mice were biopsied and tumor size was monitored following doxycycline withdrawal. Shown is a subset of tumors for which somatic loss of the wild-type p53 allele was detected by Southern analysis. Ploidy was determined on biopsy samples by analysis of DNA content by flow cytometry. Tumors showing complete regression became nonpalpable over a period of 2–3 wk following doxycycline withdrawal whereas tumors showing incomplete regression remained palpable and resumed growth within 3 wk. Mice harboring tumors that had completely regressed following doxycycline withdrawal were monitored for tumor recurrences. The time to development of either a tumor recurrence or an unrelated morbid event sacrifice is shown in parentheses. Tumor 36146 was not monitored for recurrence because a lymphoma necessitated sacrifice of the host mouse shortly after the primary mammary tumor regressed to a nonpalpable state. Tumors displaying both somatic loss of the wild-type p53 allele and aneuploidy were still capable of complete regression. N/A, not applicable.

Primary mammary tumors occurring in doxycycline-treated MTB/TWNT/p53(+/−) mice frequently gave rise to secondary doxycycline-independent tumors following withdrawal of inducer. Shown is a contingency table derived from analysis of 18 primary-secondary tumor pairs. Southern analysis was performed on genomic DNA derived from each tumor to determine whether selective loss of the wild-type p53 allele (LOH) had occurred. Eight of 18 tumor pairs were discordant for p53 LOH, and in all eight discordant pairs, p53 LOH was identified in the secondary tumor. Doxycycline-independent tumors were significantly associated with selective loss of wild-type p53 (p < 0.025, McNemar's test).

Acknowledgments

We thank Jean Richa and Gary Brown for transgene injections and Nadine Srouji and members of the Chodosh laboratory for helpful discussions and critical reading of the manuscript. This research was supported by NIH grants K08 CA79682 (E.J.G.), CA92190, CA93719, and CA94393 from the National Cancer Institute, U.S. Army Breast Cancer Research Program grant DAMD17-00-1-0401 (S.E.M.), and the University of Pennsylvania Cancer Center Core Support Grant, NCI CA16520.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Acknowledgments

Footnotes

E-MAIL ude.nnepu.dem.liam@hsodohc; FAX (215) 573-6725.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1051603.

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