Inhibition of metastasis via epithelial-mesenchymal transition by shRNA, metformin and Y27632 in breast cancer

Author(s)

Camila Leonel da Silva

• Document type: Doctoral Thesis
• Press: São José do Rio Preto. 2016-05-04.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
• Defense date: 2016-04-23
• Advisor: Debora Aparecida Pires de Campos Zuccari

Abstract

Epithelial mesenchymal transition (EMT) is the process by which cancer cells from primary tumors pass through a phenotypic conversion to invade and migrate, generating metastases in organs or tissues distant. This process can be induced by growth factors such as transforming growth factor beta (TGF-β) and its overexpression has been implicated in tumor angiogenesis, cell migration and invasion in many cancers. ROCK-1 expression is associated with the malignant character of tumors, while inhibiting this molecule results in a significant suppression of tumor metastasis. Metformin, a drug use for the treatment of diabetes, was previously shown to inhibit EMT by suppressing expression of key transcription factors in breast cancer cells. The aims were to evaluate the gene expression and protein expression of related markers metastasis, in a study in vitro and in vivo in breast cancer cell lines after treatment with metformin in addition to the gene silencing of TGF-β1 for inhibiting epithelial-mesenquimal transition. These aims were contemplated performing transfected of canine metastatic mammary tumor cell line CF41 with small interfering RNA constructs to develop clonal derivatives expressing reduced levels of TGF-β1 (TGF-β1sh cells). This was subsequently combined with metformin treatment, to look at effects on cell migration, as well as the expression of the EMT markers E-cadherin and N-cadherin, which were quantified by immunofluorescence and qRT-PCR. MCF-7 and MDA-MB-231 cell lines were treated with metformin and Y27632, after induction of EMT by TGF-β1, to examine the effects on cell migration as well as the protein expression of the ROCK-1 markers, vimentin, E-cadherin, CD44 and CD24 by immunocitochemistry. In an in vivo study, unmodified or TGF-β1 shRNA-expressing CF41 cells were injected in the inguinal region of nude athymic female mice that were treated with metformin. Mice were sacrificed after treatment and the lungs were collected to assess the number of metastases. Metastatic nodules were subsequently assessed for, N-cadherin, E-cadherin, vimentin and claudin-7 expression via immunohistochemistry. With the obtained results it was possible to assess the migration and invasion rate was lower in TGF-β1sh cells as compared to parental CF41 cells and this inhibition was significant when combined with metformin treatment. In vitro analyses demonstrated that metformin treatment reduced n-cadherin expression and increased E-cadherin expression in both CF41 and TGF-β1sh cells. After TGF-β1 induction in MDA-MB231 and MCF-7 cell lines, there was a lower protein expression of ROCK-1, vimentin, CD44 and CD24 in both cell lines after treatment with metformin and Y27632. In MDA-MB-231 cells, E-cadherin expression was increased in all treatment groups. Treatment of MDA-MB-231 cell line with metformin and Y27632 significantly reduced the invasion of these cells. In vivo studies demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-β1sh tumors. This paralleled a decreased expression of mesenchymal markers N-cadherin and vimentin, and increased expression of epithelial markers E-cadherin and claudin-7 in lung metastases.This study confirms the benefits of TGF-β1 silencing in addition to metformin and Y27632 as potential therapeutic agents in mammary tumors, by blocking EMT process and metastatic potential. (AU)

• FAPESP's process: 12/09778-1 - METASTASIS INHIBITION VIA EPITHELIAL-MESENCHYMAL TRANSITION BY RNA INTERFERENCE AND METFORMIN IN MAMMARY NEOPLASIA
• Grantee: Camila Leonel da Silva
• Support Opportunities: Scholarships in Brazil - Doctorate