We followed up a total of 105 mutant mice for two years to determine the long-term consequences of constitutive PI3K activation in the thyroid follicular cells.
After one year of age, Pten
mutant mice started showing signs of illness, and thyroid enlargement became macroscopically visible. At the end of the follow-up, we found that the female mutants had a significantly reduced lifespan(mean survival 73 weeks)compared to males(mean survival 83 weeks)(). We also analyzed the thyroid pathological features of a large group of these mice (n=54) between 8 and 12 months of age in order to further expand and validate our previously published more limited series (Yeager et al 2007
). We found that, at this age, 52% of the females had developed thyroid follicular adenomas, compared to only 12% of the males(P=0.002)(). Remarkably, analysis of mice older than one year (n=34) revealed that 50% of the females, but only 35% of the males, had developed invasive and often metastatic thyroid follicular carcinomas (see below). When combined with the incidence of follicular adenomas, 93% of the females older than one year had developed neoplastic features, compared to only 65% of the males (P=0.05)(). These data establish the thyroid-specific Pten−/−
strain as a physiologically and clinically relevant mouse model of follicular thyroid carcinoma. In addition, these mice represent a tool to further analyze the mechanisms responsible for the increased incidence of thyroid disorders in females.
Figure 1 Aging thyroid-specific Pten−/− mice develop thyroid follicular carcinomas. a, Kaplan-Meyer analysis of the gender differences in survival of mutant mice. b, prevalence of thyroid lesions in Pten mutant mice. c, d, serum levels of TSH and (more ...)
We have previously reported that the levels of TSH and T4 hormones are virtually identical in young (3-month old) wild type and mutant mice (Yeager et al 2007
). In contrast, aging mutant mice displayed TSH suppression, in most cases to levels below the detection limit of our assay (). Conversely, T4 levels were slightly but significantly increased in all adenoma-bearing mice (, black dots). These data strongly suggest that the development of thyroid neoplasia in thyroid-specific Pten−/−
mice is a cell-autonomous event, independent of TSH stimulation.
Histopathological analysis of the organs from moribund mutant mice revealed the development of follicular thyroid carcinomas with local invasion into the fibrous capsule and into the adjacent muscles and vessels (), as well as multiple lung metastases (). Neoplastic cells appeared generally well differentiated, although some areas displayed features compatible with poorly differentiated thyroid carcinomas (pleomorphic nuclei, prominent nucleoli, and a higher proliferative index)().
Figure 2 Histopathological features of thyroid carcinomas in Pten mutant mice. a, Follicular thyroid carcinoma with vascular invasion (arrow). b, multiple metastatic foci in the lungs of a Pten mutant mouse. c, magnification of the poorly differentiated tumor (more ...)
In order to characterize the effects of PI3K constitutive activation on the genes responsible for thyroid specification and function, we determined by quantitative real time PCR the expression levels of a battery of thyroid-relevant genes (). Loss of Pten, as well as neoplastic transformation of Pten−/− thyrocytes, did not significantly or consistently change the expression of the three master genes involved in the specification and differentiation of the thyroid gland, namely Foxe1, Nkx2-1, and Pax8. Hyperplastic Pten−/− thyroids had normal mRNA levels of Tshr, Duox2, Slc5a5 (NIS), and slightly reduced expression of Tpo, Tg, and Duox1. Interestingly, we found that the expression of Tpo, Duox1, and Slc5a5 was significantly higher in thyroids from female mice, irrespective of Pten status, suggesting a role for estrogen in the control of the expression of these genes. Carcinomas and, to a lesser extent, adenomas developing in Pten−/− mice showed an increase in the expression of Tshr, as well as a reduction of mRNA levels for Duox1, Duox2 and, notably, Slc5a5.
Analysis of the expression levels of several thyroid differentiation and functional markers in thyroids from male (m) and female (f) control and young mutant mice, as well as adenoma-and carcinoma-bearing female mutants.
The differences in survival and neoplastic transformation rates between mutant males and females, as well as the differences in expression levels of Tpo, Duox1, and Slc5a5, prompted us to test the hypothesis that estrogen plays a direct causal role in these gender-dependent effects. We first measured the mRNA expression of the three known estrogen receptors, ERα, ERβ, and Gpr30 in the thyroids from control and mutant mice. No significant differences were noted comparing males to females, and controls to mutants (). ERα was expressed at much higher levels than the other two receptors. Furthermore, ERα expression in isolated follicles was comparable to that in whole thyroids (data not shown), suggesting that ERα is the main estrogen receptor in the thyroid and that it is expressed by the follicular cells. Next, we ovariectomized a cohort of 4-week old control and Pten−/− immature mice (n=6) and then measured their thyrocyte proliferative index at 12 weeks of age by Ki-67 immunohistochemistry. Strikingly, complete estrogen ablation reduced the proliferative index of female thyroids to the same levels observed in mutant males (). These data provide for the first time in vivo evidence that circulating estrogens are directly responsible for the increased female susceptibility to thyroid disease, at least upon activation of the PI3K pathway. We then performed the reverse experiment: 4 week-old male mutants (n=5/group) were implanted subcutaneously with pellets for the continuous release of 17β-estradiol (0.5mg/90d) or control vehicle. After 8 weeks, thyrocyte proliferation was determined. Strikingly, estrogen treatment of mutant (but not control) males increased the thyrocyte proliferation index to the same levels observed in mutant females (). These data provide further compelling support to the direct role of estrogen in the gender-specific proliferation differences observed in Pten−/−thyroids.
Figure 4 Estrogen directly affects thyrocyte proliferation. a, expression analysis of the three estrogen receptors in thyroids from young, tumor-free mice. b, c, thyrocyte proliferation rates in mice subjected to hormonal manipulation. Ovx: ovariectomized, E2: (more ...)
Using a candidate gene approach, we searched for differentially expressed genes that might mediate the estrogen-dependent proliferation increase in female mutants. The expression level of Cdkn1b
(p27) was found higher in the thyroid, but not in the liver(used as a Pten
wild type control), of Pten−/−
male mice, compared to females (). Using Pten−/−
mouse thyroid carcinoma cell lines (Miller et al 2009
), we found that estrogen could in fact repress the expression of p27
both at the mRNA and protein level (), leading to the hypothesis that lower p27 levels in female mutants might be, at least in part, responsible for the increased thyrocyte proliferative index.
Figure 5 p27 regulation is involved in the growth-promoting effects of estrogen. a, expression analysis of p27 mRNA in the thyroid and liver from control and mutant mice. b, expression analysis of p27 mRNA in a mouse thyroid cancer cell line upon estrogen treatment. (more ...)
To test whether it is possible that the tumors developed by Pten−/− male mice spontaneously select for lower p27 expression levels, we measured p27 mRNA expression in a small series of aged male mutants (>1 year of age) that had developed hyperplastic only or neoplastic lesions (). Although the small size of the sample does not allow us to draw a final conclusion, the finding that in two out of three tumors developed by male mutants p27 expression was reduced to wild type levels support the notion that reduction of p27 in the context of PI3K activation favors tumor development.
To genetically test this hypothesis, we crossed thyroid-specific Pten−/−
mice to mice carrying a p27
null allele(Kiyokawa et al 1996
). Not surprisingly, stepwise reduction of p27 gene dosage resulted in a progressive decrease of the survival in compound mutants(). Pten−/−
mice had a mean survival of only 21 weeks, and all died by six months of age, with dramatically hyperplastic thyroids that caused dyspnea and prevented feeding. On the other hand, Pten−/−
mutants had a mean survival of 58 weeks, a 25% reduction compared to Pten−/−
mice. Strikingly, the gender differences in survival observed in Pten−/−
mice were totally rescued by the reduction of p27
gene dosage (), suggesting that p27 is a relevant mediator of the effects of estrogen on thyroid tumor incidence. In fact, when we compared the proliferative index of thyroids from young, tumor-free Pten−/−
mice, we found that loss of one p27
allele in the males resulted in a larger proliferation increase than it did in the females (). As a consequence, the gender differences in thyrocyte proliferation were drastically reduced in Pten−/−
The Pten−/−;p27+/− compound mutants survived long enough to develop adenomas and carcinomas: contrary to what we had observed in Pten−/− mice (), the incidence of neoplastic lesions was not significantly different between Pten−/−;p27+/− males and females, although a trend towards higher incidence in the latter was still detectable(). These data strongly support a role for p27 as one important mediator of estrogen action in thyroid hyperproliferation and neoplastic transformation.