Involvement of Akt, Ras and cell cycle regulators in the potential development of endometrial hyperplasia in women with polycystic ovarian syndrome

Abstract

Objective: To examine whether the abundance, localization, and/or activity of cell cycle regulators CDK2, Cyclin E, p27, and survival proteins AKT and Ras in PCOS-associated endometria (with and without hyperplasia) differ from non-PCOS endometria.

Methods: The expression of CDK2, Cyclin E, p27, AKT and Ras was measured by immunohistochemistry and/or Western blot in 9 normal endometria (NE), 12 endometria from PCOS patients without endometrial hyperplasia (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE), and 9 endometria from patients with endometrial hyperplasia (HE). The activity of CDK2 was assessed by an in vitro kinase assay.

Results: CDK2, Cyclin E and p27 proteins were expressed mainly in the endometrial epithelial cells of the studied groups. No change in the activity of CDK2 was observed in total extracts obtained from the tissue samples. However, the nuclear expression of CDK2 in epithelial cells was slightly elevated in PCOSE and significantly increased in HPCOSE when compared to NE. Higher expression of p27 was detected in the cytoplasm of epithelial cells of PCOSE and HPCOSE when compared to NE. Also, we found an increment in Ser473-AKT phosphorylation and an over-expression of the Ras oncogene in endometria of patients with PCOS.

Conclusion: The PCOS condition is associated with increased Ser473-AKT phosphorylation, elevated expression of Ras, increased cytoplasmic abundance of p27, and increased nuclear abundance of CDK2 in the endometrial epithelial cells. These biological events could potentially provide a chance for endometrial cells from PCOS patients to exit the controlled cell cycle and become hyperplastic at a later stage.

Fig. 1

The analysis of CDK2 kinase activity was performed in NE (n = 9), PCOSE (n = 12), HPCOSE (n = 7) and HE (n = 9). Cell extracts were prepared as described in Materials and methods. A portion of each extract, containing equal amounts of protein, was subjected to immunoprecipitation with anti-CDK2 antibody. Thereafter the sample was used to assay CDK2 kinase activity using histone H1 as substrate. The figure shows a representative image from the different groups of endometria studied. Endometrial tissue from patients with endometrial carcinoma (n = 3) was used as positive control [(+)].

Fig. 2

Immunohistochemical detection of CDK2 (A–D), Cyclin E (E–H) and p27 (I–L) in paraffin wax sections of endometria from women with proven fertility in the proliferative phase (NE, n = 9), untreated PCOS women (PCOSE, n = 12), PCOS women with endometrial hyperplasia (HPCOSE, n = 7), or women with endometrial hyperplasia (HE, n = 9). Positive nuclear and cytoplasmic staining was detected in epithelial and stromal cells for CDK2, Cyclin E and p27. Arrows indicate positive staining for CDK2 and Cyclin E in the nuclei of epithelial cells, and arrowheads show the staining for p27 in the cytoplasms of epithelial cells. Magnification in the panels corresponds to × 400 and the scale bar represents 10 µm. As a negative control, the primary antibody was omitted (insert in D).

Fig. 3

Semiquantitative evaluation of CDK2 (A), Cyclin E (B) and p27 (C) protein expression by HScore in epithelial cells [(nuclear compartment (NC) and cytoplasmic compartment (CC))] from the four groups studied. The values are expressed as HScore (HS) (mean ± SEM). Calculation of HScore is described in Materials and methods. *P<0.05 vs. NE.

Fig. 4

Results of Western blotting for phosphorylated AKT, total AKT, and Ras in NE, PCOSE, HPCOSE and HE. (A and C) Equal amounts of endometrial protein were loaded in each lane. p-AKT/Ser473, p-AKT/Thr308 and AKT were detected as bands with molecular mass of 60 kDa, and Ras was detected as a band with a molecular mass of 21 kDa. (B and D) p-AKT/Ser473, p-AKT/Thr308, AKT and Ras band intensities were semiquantified by scanning densitometry and normalized to β-Actin. The results are expressed as arbitrary densitometric units (AU), and the values shown are mean ± SEM in NE (n = 9), PCOSE (n = 12), HPCOSE (n = 7), and HE (n = 9). *P<0.05 in PCOSE and HPCOSE compared to NE. ^#P<0.05 in p-AKT/Ser473 vs. p-AKT/Thr308.