Deletion Of The Pten Tumor Suppressor In Pax6-expressing Surface Ectoderm Leads To Fgfr2-dependent Formation Of Periocular Epidermal Tumors

Abstract

Purpose: : Fgfr2 signaling is an important mediator of cell survival in the the lens. Upon ligand binding, Fgfr2 activates PI3 kinase (PI3K) which leads to the phosphorylation (activation) of Akt. Akt activation promotes cell survival in many cellular contexts. The activity of PI3K is antagonized by the Pten phosphatase. This led to the hypothesis that loss of Pten from the ocular surface ectoderm (ose) would rescue lens apoptosis resulting from Fgfr2 loss. While carrying out these studies we serendipitously discovered that Pten loss in the ose led to the rapid formation of periocular epidermal tumors that fail to form in the absence of Fgfr2.

Methods: : A conditional, loxP-flanked, null allele of Pten (Pten^L/L) was used to delete Pten in the ose using the Le-Cre transgenic mouse line. We also bred our Pten/Le-Cre mice to mice with a conditional deletion of Fgfr2 (Fgfr2^L/L) to obtain mice deleted for both Pten and Fgfr2 in the ose as well as Cre negative animals homozygous for the conditional deletions that were used as negative control animals. Three different genotypes were analyzed: Pten^L/L Le-Cre (Pten), Fgfr2^L/L Le-Cre (R2), and Pten^L/L Fgfr2^L/L Le-Cre (Pten/R2). Tissues were examined for morphlogy, BrdU incorporation for cell cycle analysis, and by TUNEL assay for apoptosis.

Results: : Pten mice developed tumors localized to the periocular region within 6 weeks of birth, and by 9 weeks these tumors extended to the end of the snout which is within the domain of Cre expression in Le-Cre mice. At 6 weeks, the Cre-expressing epidermis averaged 110 μm in the Pten mice, but epidermis adjacent to the Cre expressing domain remained of normal thickness (average of 16 μm in control mice). Outgrowths of abnormal cell types were also observed in the Cre expressing region, which we recognized as the tumor. The Pten mice also exhibited significantly increased proliferation in Cre-expressing epidermis. In contrast, Pten/ R2 mice did not show any external signs of epidermal tumor development, and the thickness of the epidermis as well as the proliferation rate was not increased relative to Cre negative mice. Although there was some apoptosis in the Pten epidermis, apoptosis was much more prevalent in the Pten/ R2 epidermis. Fgfr signaling appeared to be increased in the Pten mice as the Cre expressing epidermis demonstrated increased Frs2α phosphorylation. As expected, the Pten/ R2 mice exhibited reduced phosphorylation of Frs2α.

Conclusions: : Periocular epidermal cells lacking the Pten tumor suppressor gene display increased Fgfr signaling, and proliferation leading to rapid tumor development. The development of these tumors is dependent on active signaling through Fgfr2.

Keywords: apoptosis/cell death • tumors • transgenics/knock-outs