Cross-talk between PI3K and estrogen in the mouse thyroid predisposes to the development of follicular carcinomas with a higher incidence in females

Abstract

It is well known that thyroid disease is more frequent in women than in men; however, the molecular basis for this gender-based difference is still poorly understood. The activation of phosphoinositide 3-kinase (PI3K), through different mechanisms including loss of the PTEN tumor suppressor, is being increasingly recognized as a major player in the development of thyroid neoplastic lesions. Loss of Pten in the mouse thyroid results in a significant increase in the thyrocyte proliferative index, which is more prominent in the female mice. In this study, we show that 52% of the Pten(-/-) female mice, but only 12% of the males, develop follicular adenomas by 1 year of age. In addition, 50% of female mutants, but only 35% of males older than 1 year of age develop invasive, and often metastatic, follicular carcinomas. Mutant females have a significantly shorter overall survival compared with male mutants. Hormonal manipulation experiments established a direct role of estrogens in controlling the increased thyrocyte proliferation index in mutant females. Furthermore, while genetic ablation of one Cdkn1b allele accelerated the development of neoplastic lesions, it also abolished the gender differences in survival and reduced the difference in neoplastic lesion development rate, underlining a key role of p27 in mediating estrogen action in the thyroid follicular cells. These data, based on a clinically relevant model of thyroid follicular carcinoma, provide, to the best of our knowledge, for the first time in vivo evidence that circulating estrogens are directly responsible for the increased female susceptibility to thyroid disease, at least on activation of the PI3K pathway, and provide new insights into the gender-based differences characterizing thyroid neoplastic disorders.

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 T4 in aging mutant mice. Black circles in “d” correspond to mice with thyroid adenomas.

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 area indicated in “a”; note the enlarged, pleomorphic nuclei and prominent nucleoli. d, Ki-67 staining of the area shown in “c” reveals a high proliferative index.

Figure 3

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.

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: estrogen-treated, N.S.: not significant differences.

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. c, Western blot analysis of p27 levels in a mouse thyroid cancer cell line 24 hours after estrogen treatment. d, expression analysis of p27 mRNA in the thyroid from control and mutant males, as well as old (>1 year) males that had developed hyperplastic or neoplastic (Ad/Ca: Adenoma, Carcinoma) lesions. e, Kaplan-Meyer analysis showing the effect of progressive reduction of p27 gene dosage on the survival of mutant mice. f, Kaplan-Meyer analysis showing that 50% reduction of p27 gene dosage abolishes the gender-dependent differences in mutant survival. g, proliferative index (Ki-67 positive thyrocytes) of single and Pten^−/−;p27^+/− compound mutant thyroids. h, prevalence of thyroid lesions in compound mutant mice.