Cooperation between cyclin E and p27(Kip1) in pituitary tumorigenesis

Abstract

Cushing's disease is caused by glucocorticoid-resistant pituitary corticotroph adenomas. We have previously identified the loss of nuclear Brg1 as one mechanism that may lead to partial glucocorticoid resistance: this loss is observed in about 33% of human corticotroph adenomas. We now show that Brg1 loss of function correlates with cyclin E expression in corticotroph adenomas and with loss of the cell cycle inhibitor p27(Kip1) expression. Because Brg1 is thought to have tumor suppressor activity, the present study was undertaken to understand the putative contribution of cyclin E derepression produced by loss of Brg1 expression on adenoma development. Overexpression of cyclin E in pituitary proopiomelanocortin cells leads to abnormal reentry into cell cycle of differentiated proopiomelanocortin cells and to centrosome instability. These alterations are consistent with the intermediate lobe hyperplasia and anterior lobe adenomas that were observed in these pituitaries. When combined with the p27(Kip1) knockout, overexpression of cyclin E increased the incidence of pituitary tumors, their size, and their proliferation index. These results suggest that cyclin E up-regulation and p27(Kip1) loss-of-function act cooperatively on pituitary adenoma development.

Figures

Fig. 1.

Cyclin E up-regulation and loss of p27Kip1 expression in corticotroph adenomas. A, A panel of 25 human corticotroph adenomas was studied by immunohistochemistry for Brg1, cyclin E, and p27Kip1 expression. All samples were also positive for ACTH, Tpit, and GR expression. Normal pituitary tissue is always positive for nuclear Brg1 and p27Kip1 and negative for cyclin E. The upper panels represent data for adenomas that are Wt for Brg1 (i.e. positive) whereas the lower panels represent data for the Brg1-negative subset. For each, the left panel indicates percentage of tumors that are either cyclin E negative (Wt) or cyclin E positive, and the right panels indicate the proportion of each tumor group that has lost expression of p27Kip1. No tumor was ever observed to be Brg1 negative and cyclin E negative. B, Immunohistochemical analyses for a representative human Brg1-negative, cyclin E-positive, p27Kip1-negative adenoma. Normal pituitary is shown for comparison. C, The putative repressor activity of Brg1 on cyclin E expression was verified in AtT-20 cells using RNA interference with a short hairpin loop RNA target against Brg1 (shpBrg1) in comparison with a scrambled-sequence control RNA (shpCTRL). Western blot analysis of Brg1, cyclin E, and GAPDH indicated successful knockdown of Brg1 resulting in up-regulation of cyclin E. D, Sections from two representative tumors of the Brg1-negative, cyclin E-positive, and p27Kip1-negative group showing colabeling of POMC-positive cells with pHH3. All tumor samples of this group showed similar double-positive cells, but no pHH3-positive, ACTH-negative cells. IB, Immunoblotting.

Fig. 2.

Forced expression of cyclin E in mouse pituitary. A, Structure of cyclin E-expressing transgene driven by the rat POMC promoter (Tg-PCE). B, Analysis of four different transgenic lines compared with their Wt sibs for expression of Tpit, cyclin E, and GAPDH analyzed by Western blot of adult pituitary extracts. The number on top refers to the identification number of each transgenic line. Line 337 was mostly used for further studies, but line 649 also produced similar results. C, Characterization of embryonic d 18.5 (e18.5) Tg-PCE transgenic mice. Expression of the transgene was assessed on pituitary sections by immunofluorescence. Cyclin E overexpression is detected in a large number of IL melanotroph cells as well as in some AL cells (D), whereas no cyclin E was detected in the ACTH-positive cells of sib control embryos (C). Analysis of pHH3-positive cells showed few positive cells (usually POMC-negative; E, inset) for this marker of mitosis in control pituitary (E) but much more frequent positive cells in the transgenic pituitary (F), including cells that are positive for both pHH3 and ACTH (F, inset).

Fig. 3.

Cyclin E expression increases pituitary proliferation index. A, Cohorts of Tg-PCE mice and their control sibs (Wt) were investigated at 2 yr of age for a pituitary phenotype. Six transgenic pituitaries of a total of 17 were found to have abnormal pituitaries, three with IL hyperplasia (C) and three with AL adenomas (D–F). B, Quantitation of cyclin E and Ki67-positive cells in adult ILs of Tg-PCE transgenic pituitaries compared with their Wt control sibs. Cyclin E-positive and Ki67-positive (a marker of proliferation) cells were identified by immunohistochemistry on pituitary sections of 8-month-old and 2-yr-old mice as indicated. For each group, the number of positive cells was counted on duplicate sections from eight to 10 different mice. F, Summary of marker expression in the three AL adenomas. Only one tumor was positive for the differentiation markers Pit-1, PRL, and GH.

Fig. 4.

Cyclin E overexpression causes centrosome abnormalities. Centrosomes (C) revealed using γ-tubulin immunofluorescence (19 ) were analyzed on two sections from each mouse reported in Fig. 3A; analyses were only performed on IL tissues with normal appearance, i.e. not in hyperplastic or tumor areas. Panel A, Dividing cells have two visible centrosomes (C = 2), and abnormal numbers of centrosomes are reported as C ≥ 3. Each dot represents data for a different mouse. Bar represents the means ± sem. Panel B, Quantitation of structural centrosome abnormalities. These were never observed in control tissues. Panel C, Examples of centrosomes observed in this study.

Fig. 5.

Effect of Brg1 knockdown [small interfering RNA (siRNA)] in AtT-20 cells on cyclin E and p27Kip1 mRNA and protein levels. A, Quantitative real-time PCR (RT-QPCR) analysis of Brg1, cyclin E, and p27Kip1 transcripts in AtT-20 after Brg1 knockdown. Brg1 transcripts are decreased 3.1-fold (P ≤ 0.0001) whereas cyclin E and p27Kip1 transcripts are increased 1.7-fold (P ≤ 0.005) and 2.8-fold (P ≤ 0.05), respectively (n = 3). B, Levels of Brg1, cyclin E, p27Kip1, phospho-p27Kip1 (Thr187), and GAPDH in extracts from AtT-20 cells treated with siCTRL and siBrg1 for 24 and 48 h revealed by Western blot. IB, Immunoblotting.

Fig. 6.

Cyclin E cooperates with p27Kip1 loss of function for pituitary tumor development. A, Photographs of representative in sellae pituitaries from 8-month-old mice: Wt, Tg-PCE and p27Kip1 heterozygote (Tg-PCE;p27Kip1+/−), p27Kip1 knockout (p27Kip1−/−), and Tg-PCE transgenic p27Kip1 knockout (Tg-PCE;p27Kip1−/−). B, Distribution of pituitary weights for mice of the different genotypes. The number of pituitaries in each group is indicated in Table 1. Bars represent the medians. C, Colabeling of representative anterior pituitary sections for mice of the indicated genotypes using Ki67 as marker of proliferating cells and ACTH to identify corticotrophs. D, Quantitation of Ki67-positive cells in the IL and AL of pituitaries from each genotype. The number of mice studied in each group is indicated at the bottom and for each mouse, two sections were quantitated. In each case, the percentage of Ki67-positive cells was subdivided into four categories as indicated. The increased Ki67 index observed in AL of Tg-PCE;p27Kip1−/− compared with p27Kip1−/− pituitaries is statistically significant (P ≤ 0.03).