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Tumor biomarkers in liquid biopsy: study of resistance mechanisms to block DNA repair: a comparative analysis

Grant number: 17/15006-5
Support type:Regular Research Grants
Duration: March 01, 2018 - February 29, 2020
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Pathology
Principal Investigator:Debora Aparecida Pires de Campos Zuccari
Grantee:Debora Aparecida Pires de Campos Zuccari
Home Institution: Faculdade de Medicina de São José do Rio Preto (FAMERP). Secretaria de Desenvolvimento Econômico (São Paulo - Estado). São José do Rio Preto , SP, Brazil
Assoc. researchers:Heidge Fukumasu

Abstract

Breast cancer (BC) is the most prevalent type of tumor among women and has a high mortality rate. This is attributed to failure to detect early disease and absence of effective markers to estimate the risk of cancer progression and to predict tumor response to chemo and radiotherapy. In dogs, being about 50% of the tumors diagnosed as malignant. In addition, BC in dogs shares many similarities with these tumors in humans, which makes them excellent models for the study of cancer biology as well as for testing therapeutic agents. BC is characterized by high intratumoral heterogeneity, so called due to the coexistence of different clones of tumor cells in the same tumor. This process of clonal evolution occurs due to the sequential acquisition of mutations culminating in drug resistance throughout the exposure to the treatment, which demonstrates the necessity of a personalized therapeutic approach for each patient. Currently, studies are beginning to investigate methods of early diagnosis of cancer and follow the response to treatment by detecting circulating cells (CTCs) and DNA fragments (ctDNA) released by the primary tumor or its metastases in the blood. This innovative procedure is known as liquid biopsy and is so called because it only requires the collection of blood or other body fluids, thus avoiding invasive procedures such as conventional tissue biopsies. It allows access to considerable tumor information that guides the patient's prognosis and dynamic disease monitoring in real time during the follow-up period. In this way, searching the mechanisms involved in chemoresistance to eliminate it has become a proposal of great interest and an innovative alternative is the use of PARP inhibitors (PARPi). Poly (ADP-ribose) polymerase (PARP) is a nuclear protein that participates in the DNA repair mechanism due to single-stranded DNA changes (SSB) in the same way that BRCA-1 and BRCA-2 genes are responsible for the repair of double-stranded DNA (DSB). Both mechanisms have the aim of maintaining the genomic integrity of the cells, thereby reducing the sensitivity of tumor cells to chemotherapeutic agents. Thus, one objective of this study is to verify the presence of mutations by liquid biopsy as well as to qualify them in the primary tumor and to test the sensitivity to specific drugs in organoid culture. A second objective is to use the PARPi associated with different chemotherapeutic agents, analyzing its correlation with Schlafen 11 gene (SLFN11) and Angiotensin II Receptor Type 1 (AGTR-1), and the mutations that originated after chemoresistance in human breast cancer and canine mammary neoplasia. We will collect liquid biopsies for ctDNA extraction to analyze the panel of mutations by Next Generation Sequencing (NGS). Cell lines and primary culture derived from tumor fragments obtained by surgical excision will be cultured in organoids and treated with different chemotherapeutics to evaluate the specific response, followed by chemoresistance and treatment in association with PARPi, losartan and subjected to the CRISP/Cas9 technique for overexpression of SLFN11 gene and underexpression of AGTR-1. At the end, the results of the liquid biopsy will be analyzed and its ability to detect mutations, and the correlation between the different mutations and the responsiveness and resistance of the tumor cells to the chemotherapeutic will be defined as an attempt to determine a personalized therapeutic protocol for mammary tumor patients. In addition, from these data, it will be possible to understand the mechanisms of action of prolonged treatments, allowing greater chances of remission for the patient with breast cancer. (AU)