35 Decreased Epithelial Mesenchymal Transition Process After AGTR-1 Gene Edition By Crispr/Cas9, Losartan, and PARP Inhibitor Treatment In Breast Cancer Cell Line

Marina Gobbe Moschetta-Pinheiro, PhD¹ ; Jucimara Colombo¹ ; Julia Ferreira Balan¹ ; Bianca Carlos Nascimento¹ ; Murilo de Souza Tuckumantel¹ ;Debora Aparecida Pires de Campos Zuccari¹ .

¹ Laboratório de Investigação Molecular no Câncer, Faculdade de Medicina de São José do Rio Preto, FAMERP, São José do Rio Preto, SP, Brazil.

Background

Tumor progression after chemotherapy remains a major obstacle to successful treatment of cancer. Among the mechanisms of chemoresistance of tumors, we highlight the ability of cells to repair DNA after chemotherapy, leading to the epithelial-mesenchymal transition (EMT) process, resulting in metastases. Olaparib (Lynparza) is a PARP inhibitor (PARPi) able to block DNA repair and has been studied as an alternative to prevent the chemoresistance of tumor cells. Losartan, in turn, binds to the receptor AGTR-1, inhibiting the process of angiogenesis in breast cancer, consequently decreasing the formation of metastases. In this context, the objectives of this work were to evaluate the efficacy of olaparib and losartan as a therapeutic strategy in breast tumors as well as to analyze EMT markers after the process of AGTR-1 gene editing by CRISPR/Cas9.

Materials and Methods

Breast cancer cell line (MCF-7) was cultured in a high-glucose culture, Dulbecco’s Modified Eagle’s Medium, at 37°C in 5% CO2. The cells were then treated with PARPi and losartan or submitted to the CRISPR/Cas9 technique for inhibition of the AGTR-1 receptor. After 24 hours, we used the immunofluorescence technique to analyze protein expression and real-time polymerase chain reaction for gene expression of EMT markers. In addition, the cells’ invasion capacity was also verified after AGTR-1 inhibition by CRISPR/Cas9 technique.

Results

The N-cadherin, vimentin, and PARP-1 protein and gene expression significantly decreased after treatment with PARPi or losartan (P <.05) compared with the control group (without treatment). There was still a decrease of cell migration ability after PARPi or losartan treatment (P <.05) and the same was observed after AGTR-1 gene edition by CRISPR/Cas9 technique (P <.05).

Conclusions

The results showed that PARPi and losartan act in the regulation of EMT process in breast cancer cells.

Financial Support: FAPESP