Inhibitors of SCF-Skp2/Cks1 E3 Ligase Block Estrogen-Induced Growth Stimulation and Degradation of Nuclear p27kip1: Therapeutic Potential for Endometrial Cancer

Abstract

In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3μM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%-62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

S-phase kinase-associated protein 2 expression interference inhibits breast cancer cell proliferation

“…Our results showed that SKP2 expression is significantly higher in patients with invasive breast cancer than in normal breasts. Previous research has suggested that SKP2 mainly degrades CKI p27 in the cell cycle, while high expression of SKP2 in malignant tumors is often accompanied by significantly reduced p27 (Pavlides et al, 2013;Chan et al, 2014). This is in agreement with our results.…”

Roles of F-box proteins in cancer

“…With a similar mechanism, SKP2 E3 ligase inhibitors (SKP2E3LIs), which are another group of inhibitors that block the interaction of SKP2 with p27, efficiently hindered the oestrogen-induced stimulation of cell growth and the degradation of nuclear p27 (REF. 155). …”

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

“…SR microscopy circumvents the conventional resolution limit of light microscopy by temporally separating emitting fluorophores and computationally fitting the location of each below the diffraction limit. Reconstructing thousands of points in this manner generates an image with a resolution typically an order of magnitude better that that of conventional microscopy (32)(33)(34). In addition to this approach, we used smFRET, a powerful method capable of monitoring the dynamics of individual nucleoprotein complexes in real time (35).…”