In vivo and in vitro functional validation of the Sonic Hedgehog signaling pathway inhibitors in uterine leiomyoma and leiomyosarcoma

Abstract

Leiomyoma and leiomyosarcoma are mesenchymal tumors that occur in the uterus. These tumors have variable clinical behavior, compromising fertility and may even lead to death. Leiomyoma is a benign tumor commonly found in reproductive age women. Since leiomyosarcoma is a rare neoplasm comprising nearly 40% of the uterine sarcomas. Both leiomyosarcoma and leiomyomas are myometrial neoplasms that display the same pattern of cell differentiation, but with completely different clinical progression. Several studies have shown that aberrant activation of the Sonic Hedgehog signaling pathway (SHH) is related to several types of cancer development, because it plays important role in the cell proliferation and differentiation. In a previous study, our group evaluated the expression profile of SHH pathway molecules in uterine mesenchymal tumors. The SMO protein, GLI1 and SHH was found with higher expression in leiomyosarcomas, followed by leiomyomas and negative in adjacent normal myometrium. These results aroused our interest in evaluating the therapeutic potential of the SHH pathway inhibitors in these tumors, because very promising results were obtained in this sense for other cancers. Thus, this project aim is to evaluate, in vitro and in vivo, the action of Sonic Hedgehog inhibitors in leiomyoma and leiomyosarcoma cells. To do this, initially, specific inactivation of the SHH pathway proteins will be performed in cell lines by RNAi and treatment with specific inhibitors of SMO (GDC0449, LDE225 and cyclopamine); GLI 1 (GANT56, GANT61, IPH1 and 6) and SHH (Robotinikinin). After the treatments, the cells will be checked for the targets inhibition and evaluated as to their migration and invasion capacity. Also, from the previous step, the most effective inhibitors will be tested in primary culture of cells from patients with leiomyoma and leiomyosarcoma. After data analysis, we will select up to three inhibitors to assess their effects in vivo. We will evaluate 20 female mice divided into 5 groups containing 4 animals by inhibitor tested (G1 control group - vehicle only; G2, animals inoculated with leiomyoma cells; G3, animals inoculated with leiomyoma cells and threated with inhibitor; G4, animals inoculated with leiomyosarcoma cells; G5, animals inoculated with leiomyosarcoma and threated with inhibitor). The experiment will be performed in duplicate and we will to evaluate the effects of the treatment (toxicity), the size of tumors and expression of target genes and proteins. The effectiveness of inhibitors in cells grown and induced tumors will be evaluated by Western Blott and real time PCR for the pathway molecules (SHH, PTCH1, SMO, Sufu and GLIs 1-3) and for their downstream targets (BMP4, BCL-2 and CCND1). All results will be submitted to statistical analysis. (AU)