Targeting Hedgehog pathway and DNA methyltransferases suppress the phenotype of uterine leiomyosarcoma cells

Abstract Background: Uterine leiomyosarcoma (LMS) is an aggressive tumor with poor prognosis, high rates of recurrence and metastasis. Several studies have shown an essential role of Hedgehog (HH) signaling in tumor development. The role of HH pathway in LMS is still unclear. Methods: Uterine smooth muscle (USTM) and LMS cells lines were used to evaluate the mRNA and protein expression of HH components. MTT assay was performed to evaluate the inhibitory effect of SMO, GLI1 and DNMT inhibitors for proliferation. Nuclear and cytoplasm fractions were prepared. Protein and RNA expression levels were determined by Western blot and q-PCR analysis. Migration, invasion and apoptosis assays were performed to determine the LMS phenotype after the treatments. The statistical analysis was performed using GraphPad Prism 5, and the statistical significance was accepted for p<0.05.Results: LMS analyses showed upregulation of SMO and GLIs (1, 2 and 3) in LMS compared to the myometrium and uterine leiomyoma. Increased nuclear translocation of GLIs proteins was found in LMS cells as well. After that, we hypothesized that HH pathway could be activated in uterine LMS and its blocking would lead to LMS malignant phenotype suppression. Inhibition of SMO and GLI after treatment with LDE225 and Gant61 respectively, induced both protein downregulation as well as decreased GLIs nuclear translocation. Also, abnormal increased expression of DNMT (1, 3a, and 3b) was observed in LMS, and treatment with a DNMT inhibitor decreased both DNMTs expression and GL1 nuclear translocation. Conclusion: Our analyses showed that deactivation of SMO, GLI and DNMTs was able to inhibit LMS phenotype. Importantly, the combination of those treatments exhibited a potentiated effect on LMS malignant phenotype suppression, leading to a deactivating HH pathway. In conclusion, our studies may provide novel options for uterine LMS patients´ target therapy.