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. 2003 Feb 15;17(4):488-501.
doi: 10.1101/gad.1051603.

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

Edward J Gunther  1 , Susan E Moody, George K Belka, Kristina T Hahn, Nathalie Innocent, Katherine D Dugan, Robert D Cardiff, Lewis A Chodosh
Affiliations

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

Edward J Gunther et al. Genes Dev. .

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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Figures

Figure 1
Figure 1
Doxycycline-dependent activation of Wnt signaling in MTB/TWNT mice. (A) Schematic depicting the strategy to permit doxycycline-dependent expression of Wnt1. Bitransgenic mice carrying both the MMTV-rtTA-pA (MTB) and TetO–Wnt1 (TWNT) transgenes express the rtTA chimeric transcription factor in the mammary epithelium but do not express the bicistronic message encoded by the independently integrated TWNT transgene driven by a minimal CMV promoter containing multimerized tet operator sequences. In the presence of inducer, rtTA binds doxycycline and undergoes a conformational change that facilitates recognition of tet operator sequences and activation of the TWNT promoter. (B) Doxycycline-dependent expression of Wnt1 in the mammary glands of MTB/TWNT mice. Northern hybridization analysis of total RNA from the mammary glands of 5-week-old nulliparous bitransgenic MTB/TWNT female mice. Mice were either left untreated or administered 2 mg/mL doxycycline in drinking water for 96 h. Note coordinate up-regulation of c-myc, a known transcriptional target of canonical Wnt signaling, and the Wnt1 transgene. (C) Doxycycline-dependent mammary ductal side-branching and hyperplasia in MTB/TWNT mice. Mammary glands harvested from MTB/TWNT and wild-type FVB/N control mice after treatment with doxycycline (Dox) as indicated were spread on slides, fixed, and subjected to carmine staining.
Figure 2
Figure 2
Mammary tumorigenesis in chronically induced MTB/TWNT mice. (A) Tumor incidence curves for transgenic mice. A cohort of 52 female bitransgenic MTB/TWNT mice was administered 2 mg/mL doxycycline beginning at 4–6 wk of age and monitored twice weekly for tumor formation. Tumors arose with a median latency of 20 wk of doxycycline induction. No tumors arose in control cohorts that included doxycycline-induced monotransgenic mice (MTB, n = 20; TWNT, n = 15) and uninduced bitransgenic MTB/TWNT mice (n = 9). (B) MTB/TWNT mice develop mammary adenocarcinomas. H&E sections from mammary tumors arising in doxycycline-induced MTB/TWNT mice. The middle panel depicts an area of squamous differentiation seen in a subset of MTB/TWNT tumors.
Figure 3
Figure 3
Regression of mammary tumors and down-regulation of Wnt signaling following doxycycline withdrawal. (A) Regression of primary mammary tumors in MTB/TWNT mice. The size of biopsy-proven mammary tumors arising in chronically induced MTB/TWNT mice was monitored following doxycycline withdrawal. Shown are representative regression curves for five independent tumors; 33 of 35 (94%) tumors analyzed regressed to a nonpalpable state. (B) Prompt down-regulation of luciferase activity in MTB/TWNT mammary tumors. Mammary tumors were explanted onto the flanks of doxycycline-treated female mice, and tumor outgrowths were harvested from hosts maintained on doxycycline and hosts from whom doxycycline had been withdrawn for the indicated times. A representative analysis performed on grafts derived from a single tumor is shown, with duplicate grafts at each time point. (C) Down-regulation of Wnt signaling in MTB/TWNT mammary tumors. Northern hybridization analysis of total RNA from MTB/TWNT tumor explants. Wnt1 transgene expression is down-regulated within 18 h (top two panels) but remains detectable until 54 h (second panel from top, 7-d exposure). Down-regulation of two known transcriptional targets of canonical Wnt signaling, c-myc and Tcf7, occurs in response to abrogation of Wnt1 transgene expression. Continued expression of rtTA at 54 and 96 h post-doxycycline withdrawal demonstrates that down-regulation of Wnt1 transgene, c-myc, and Tcf7 is not solely a consequence of loss of tumor cells or tumor cell viability.
Figure 4
Figure 4
Regression of lung metastases following doxycycline withdrawal. Lung metastases (n = 7) arising in three independent MTB/TWNT mice were explanted onto the flanks of wild-type FVB/N hosts maintained on doxycycline. The primary mammary tumor presumed to be the source of each metastasis was grafted onto the contralateral flank of each host mouse. (A) The histology of H&E-stained sections derived from lung metastases closely resembled that of primary tumors, both in the case of in situ metastases (middle panel) and explanted metastases (right panel). (B) Similar rate and extent of regression for explants derived from primary mammary tumors and lung metastases. Regression curves for duplicate explants derived from a representative primary-metastasis pair.
Figure 5
Figure 5
Effect of p53 loss-of-function on the occurrence of tumor aneuploidy and tumor regression following abrogation of Wnt signaling. (A) Regression of MTB/TWNT tumors in the absence of p53. The size of biopsy-proven mammary tumors arising in chronically induced MTB/TWNT/p53(−/−) mice (n = 7) was monitored following doxycycline withdrawal. Regression curves for three representative tumors are shown. Tumors lacking p53 by germline transmission of two null alleles regressed with similar kinetics as tumors arising in mice with wild-type p53 alleles (cf. Figs. 5A and 3D). Tumor recurrence could not be assessed in p53(−/−) animals due to the rapid occurrence of lymphomas. (B) p53 loss of heterozygosity (LOH) in a subset of tumors arising in MTB/TWNT/p53(+/−) mice. Southern hybridization analysis was performed on genomic DNA derived from mammary tumors arising in mice inheriting a single null allele for p53. Selective loss of the wild-type p53 allele was apparent in a subset of these mammary tumors (lanes marked with *). (C) Correlation of tumor aneuploidy with p53 LOH. The ploidy of tumors arising in MTB/TWNT/p53(+/−) mice was determined by flow cytometric analysis of propidium iodide-stained nuclei. Representative DNA histograms for tumors without (left panels) and with (right panels) detectable p53 LOH by Southern analysis are shown. Aneuploidy was common in tumors with detectable p53 LOH (6 of 7 tumors, 86%), but rare in tumors without detectable p53 LOH (1 of 17 tumors, 6%). (D) Incomplete regression and regrowth of a subset of tumors arising in MTB/TWNT/p53(+/−) mice. The size of biopsy-confirmed mammary tumors arising in MTB/TWNT/p53(+/−) mice was monitored following doxycycline withdrawal as above. Regression curves for six representative tumors are shown. Unlike tumors arising in MTB/TWNT/p53(+/+) mice, which only rarely failed to regress completely following doxycycline withdrawal (2 of 35 tumors, 6%; see Fig. 3D), a substantial fraction of tumors arising in MTB/TWNT/p53(+/−) mice regressed incompletely following doxycycline withdrawal and resumed growth (12 of 30 tumors, 40%).
Figure 6
Figure 6
Decreased tumor-free survival in p53 heterozygous mice. (A) Accelerated recurrence of mammary tumors in p53 heterozygous vs. p53 wild-type mice. MTB/TWNT mice that had biopsy-proven mammary tumors that had regressed to a nonpalpable state following doxycycline withdrawal were monitored for the development of doxycycline-independent tumors for periods up to 1 yr. Recurrence of mammary tumors was significantly accelerated in p53 heterozygous vs. p53 wild-type mice (p < 0.0001, log-rank test). (B) p53 LOH in a subset of doxycycline-independent recurrent mammary tumors arising in MTB/TWNT/p53(+/−) mice. Southern hybridization analysis was performed on genomic DNA derived from paired primary mammary tumors (P) and doxycycline-independent recurrent mammary tumors (R) arising in mice inheriting a single null allele for p53. In a subset of primary-recurrence tumor pairs, the recurrent tumor demonstrated selective loss of the wild-type p53 allele. Note that the tumor recurrence could not be assessed in p53(−/−) mice due to the occurrence of lymphomas.
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