Activated mutant p110α causes endometrial carcinoma in the setting of biallelic Pten deletion

Abstract

PTEN and PIK3CA mutations occur with high frequency in uterine endometrioid carcinoma (UEC). Although PTEN mutations are present in complex atypical hyperplasia and carcinoma, PIK3CA mutations are restricted to carcinoma. We generated mouse models harboring Pten loss and/or activated Pik3ca in the endometrial epithelium to investigate their respective roles in the pathogenesis of UEC. Presence of an activated mutant Pik3ca on the background of Pten loss led to aggressive disease, with 100% of mice exhibiting carcinoma. Expression of Pik3ca with E545K mutation alone was unable to cause hyperplasia or cancer in the uterus and did not activate Akt as effectively as Pten deletion in short-term cultures of mouse endometrial epithelium, likely explaining the lack of phenotype in vivo. We also report that nuclear localization of FOXO1 correlated with PTEN mutational status irrespective of the PIK3CA status in endometrial cancer cell lines. Furthermore, gene expression profiles resulting from Pten loss or activation of Pik3ca in primary mouse endometrial epithelial cells exhibit minimal overlap. Thus, Pten and Pik3ca have distinct consequences on the activation of the phosphatidylinositol 3-kinase pathway in endometrial epithelium and are likely to affect other nonoverlapping cellular mechanisms involved in the development and progression of the most common type of uterine cancer.

Figure 1

Photomicrographs of hematoxylin and eosin (H&E)–stained mouse uteri. A and B: Complex atypical hyperplasia (CAH; A) and hemorrhagic cysts (B) in Wnt-Cre^+/−;Pten^f/+;Pik3ca^E545K uteri at 5 months of age. C: Low-magnification H&E image of Ksp-Cre^+/−;Pten^f/f mice with CAH involving entire luminal and glandular epithelium. D: High-magnification image of CAH with areas of squamous metaplasia. E and F: Low-magnification (E) and high-magnification (F) image of Ksp-Cre^+/−;Pten^f/f;Pik3ca^E545K uteri with carcinoma and myometrial invasion. G: Gross morphological features of Ksp-Cre^+/−;Pten^f/f;Pik3ca^E545K uteri with both ovaries engulfed in cysts. H: Gross morphological features of Ksp-Cre^+/−;Pten^f/f mice exhibiting uterine disease but normal ovaries. I: Low-magnification H&E image of Ksp-Cre^+/−;Pik3ca^E545K uterus with normal histological features. J: High-magnification image of I. Original magnifications: ×200 (A, D, F, and J); ×40 (B, C, E, and I).

Figure 2

Recombination of mutant Pik3ca allele and activation of Akt in primary cultures. A: Agarose gel electrophoresis of PCR analysis of DNA extracted from microdissected, formalin-fixed, paraffin-embedded tissue from Ksp-Cre^+/−;Pik3ca^E545K (lane 1), Ksp-Cre^+/−;Pten^f/+;Pik3ca^E545K (lane 2), Ksp-Cre^+/−;Pten^f/f (lane 3), and Ksp-Cre^+/−;Pten^f/f;Pik3ca^E545K (lane 4) mice. B and C: p-Akt immunohistochemical analysis of uterine sections of Ksp-Cre^+/−;Pten^f/+;Pik3ca^E545K (B) and Ksp-Cre^+/−;Pik3ca^E545K (C) mice. D: Immunoblot analysis of whole cell extracts of Adeno-GFP or Adeno-Cre–treated primary uterine epithelial cells isolated from Pten^f/f, Pik3ca^E545K, and Pten^f/f;Pik3ca^E545K mice using Pten, p-Akt, Akt, and actin antibodies. E: Quantitation of p-Akt in D. Graph represents p-Akt/Akt ratios from three independent experiments. Data represent means ± SD. GFP, green fluorescent protein; WB, water blank.

Figure 3

Immunohistochemical (IHC) analysis of downstream signaling molecules. A and B: Positive IHC staining with p-Gsk3β (A) and p-Pras40 (B) antibodies in uteri with complex atypical hyperplasia (CAH; arrow) as compared to normal epithelium (arrowheads). C: Loss of nuclear FoxO1 staining in CAH (arrows) compared to the intense nuclear staining of normal epithelium (arrowheads). D: Loss of nuclear FoxO1 in carcinoma. E: Immunoblot analysis depicting subcellular localization of FOXO1 in endometrial cancer cell lines. Lamin A/C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as controls to determine relative purity of nuclear (N) and cytoplasmic (C) extracts. WT, wild type.

Figure 4

RNA sequencing analysis. A: Venn diagram depicting the differentially expressed genes (DEGs) common and/or unique to Pten^f/f, Pik3ca^E545K, and Pten^f/f;Pik3ca^E545K. B: Real-time quantitative PCR analysis to validate expression of DEGs identified by RNA sequencing. Graph depicts fold change in expression of Axin2, Hspa1a, and Cdkn2a in Pten^f/f, Pik3ca^E545K, and Pten^f/f;Pik3ca^E545K cells as compared to green fluorescent protein (GFP)–treated cells. Means ± SEM are summarized. N = number of independent experiments performed for each gene.