Targeting Hedgehog Pathway and DNA Methyltransferases in Uterine Leiomyosarcoma Cells

Abstract

Uterine leiomyosarcoma (LMS) is an aggressive tumor that presents poor prognosis, high rates of recurrence and metastasis. Because of its rarity, there is no information available concerning LMS molecular mechanisms of origin and development. Here, we assessed the expression profile of Hedgehog (HH) signaling pathway markers and the effects of their pharmacological inhibition on uterine smooth muscle (UTSM), leiomyoma and LMS cells. Additionally, we also evaluated the effects of DNMTs inhibition on LMS cells behavior. Cell proliferation, migration and apoptosis rates were evaluated by MTT, Scratch and Annexin V assays, respectively. RNA expression and protein levels were assessed by qRT-PCR and Western blot. We found that SMO and GLIs (1, 2 and 3) expression was upregulated in LMS cells, with increased nuclear levels of GLI proteins. Treatment with LDE225 (SMOi) and Gant61 (GLIi) resulted in a significant reduction in Glis protein levels in LMS (p < 0.05). Additionally, the expression of DNMT (1, 3a, and 3b), as well as GLI1 nuclear expression, was significantly decreased after treatment with HH inhibitor in LMS cells. Our results showed that blocking of SMO, GLI and DNMTs is able to inhibit LMS proliferation, migration and invasion. Importantly, the combination of those treatments exhibited a potentiated effect on LMS malignant features due to HH pathway deactivation.

Keywords: DNA methyltransferases; hedgehog signaling; inhibitor; uterine leiomyosarcoma.